Costimulation of chimeric antigen receptors by myd88 and cd40 polypeptides

ABSTRACT

The technology relates generally to the field of immunology and relates in part to methods for activating T cells and other cells resulting in an immune response against a target antigen. The technology also relates to costimulation of therapeutic cells that express chimeric antigen receptors that recognize target antigens using chimeric MyD88- and CD40-derived polypeptides. The technology further relates in part to therapeutic cells that express chimeric antigen receptors, wherein the chimeric antigen receptors have an endodomain that includes MyD88- and CD40-derived polypeptides, and methods for treating patients using the modified therapeutic cells.

RELATED APPLICATIONS

Priority is claimed to U.S. Provisional Patent Application Ser. No.62/044,885, filed Sep. 2, 2014, entitled “Costimulation of ChimericAntigen Receptors by MyD88 and CD40 Polypeptides,” to U.S. ProvisionalPatent Application Ser. No. 62/115,735, filed Feb. 13, 2015, entitled“Costimulation of Chimeric Antigen Receptors by MyD88 and CD40Polypeptides,” and to U.S. Provisional Patent Application Ser. No.62/143,503, filed Apr. 6, 2015, entitled “Costimulation of ChimericAntigen Receptors by MyD88 and CD40 Polypeptides.” The entire content ofthe foregoing applications are incorporated herein by reference in theirentireties, including all text, tables and drawings, for all purposes.

FIELD

The technology relates generally to the field of immunology and relatesin part to methods for activating T cells and other cells resulting inan immune response against a target antigen. The technology also relatesto costimulation of therapeutic cells that express chimeric antigenreceptors that recognize target antigens using chimeric MyD88- andCD40-derived polypeptides. The technology further relates in part totherapeutic cells that express chimeric antigen receptors, wherein thechimeric antigen receptors have an endodomain that includes MyD88- andCD40-derived polypeptides, and methods for treating patients using themodified therapeutic cells.

BACKGROUND

T cell activation is an important step in the protective immunityagainst pathogenic microorganisms (e.g., viruses, bacteria, andparasites), foreign proteins, and harmful chemicals in the environment,and also as immunity against cancer and other hyperproliferativediseases. T cells express receptors on their surfaces (i.e., T cellreceptors) that recognize antigens presented on the surface of cells.During a normal immune response, binding of these antigens to the T cellreceptor, in the context of MHC antigen presentation, initiatesintracellular changes leading to T cell activation. Chimeric antigenreceptors (CARs) are artificial receptors designed to convey antigenspecificity to T cells without the requirement for MHC antigenpresentation. They include an antigen-specific component, atransmembrane component, and an intracellular component selected toactivate the T cell and provide specific immunity. Chimeric antigenreceptor-expressing T cells may be used in various therapies, includingcancer therapies. Costimulating polypeptides may be used to enhance theactivation of CAR-expressing T cells against target antigens, andtherefore increase the potency of adoptive immunotherapy.

SUMMARY

Transduced or transfected T cells and other cells may express a chimericantigen receptor, resulting in activation of T cell immunity in thepresence of a target antigen. Chimeric antigen receptors (CARs) areartificial receptors designed to convey antigen specificity to T cells.They generally include an antigen-specific component, a transmembranecomponent, and an intracellular component selected to activate the Tcell and provide specific immunity. Chimeric antigen receptor-expressingT cells may be used in various therapies, including cancer therapies.

The chimeric antigen receptor polypeptides may include endogenoussignaling or activation domains to increase the potency of the chimericantigen receptor modified cell. In other examples, the signaling oractivation domains may be incorporated into a separate polypeptide, achimeric stimulating molecule, which may be co-expressed with a chimericantigen receptor, for example, a first-generation CAR, in the modifiedcell. T cell activation may be observed, for example, by the expressionand secretion of inflammatory cytokines and chemokines. The activatedcells may be used to increase the immune response against a disease, orto treat cancer by, for example, reducing the size of a tumor. Thetherapeutic course of treatment may be monitored by determining the sizeand vascularity of tumors by various imaging modalities (e.g. CT, bonescan, MRI, PET scans, Trofex scans), by various standard bloodbiomarkers (e.g. PSA, circulating tumor cells (CTCs)), or by serumlevels of various inflammatory, hypoxic cytokines, or other factors inthe treated patient.

In some therapeutic instances, a patient might experience a negativesymptom during therapy using chimeric antigen receptor-modified cells.In some cases these therapies have led to side effects due, in part, tonon-specific attacks on healthy tissue. Therefore, in some embodimentsare provided nucleic acids, cells, and methods wherein the modified Tcell also expresses an inducible Caspase-9 polypeptide. If there is aneed, for example, to reduce the number of chimeric antigen receptormodified T cells, an inducible ligand may be administered to thepatient, thereby inducing apoptosis of the modified T cells.

The antitumor efficacy from immunotherapy with T cells engineered toexpress chimeric antigen receptors (CARs) has steadily improved as CARmolecules have incorporated additional signaling domains to increasetheir potency. T cells transduced with first generation CARs, containingonly the CD3ζ intracellular signaling molecule, have demonstrated poorpersistence and expansion in vivo following adoptive transfer (Till B G,Jensen M C, Wang J, et al: CD20-specific adoptive immunotherapy forlymphoma using a chimeric antigen receptor with both CD28 and 4-1 BBdomains: pilot clinical trial results. Blood 119:3940-50, 2012; Pule MA, Savoldo B, Myers G D, et al: Virus-specific T cells engineered tocoexpress tumor-specific receptors: persistence and antitumor activityin individuals with neuroblastoma. Nat Med 14:1264-70, 2008; Kershaw MH, Westwood J A, Parker L L, et al: A phase I study on adoptiveimmunotherapy using gene-modified T cells for ovarian cancer. ClinCancer Res 12:6106-15, 2006), as tumor cells often lack the requisitecostimulating molecules necessary for complete T cell activation. Secondgeneration CAR T cells were designed to improve proliferation andsurvival of the cells. Second generation CAR T cells that incorporatethe intracellular costimulating domains from either CD28 or 4-1 BB(Carpenito C, Milone M C, Hassan R, et al: Control of large, establishedtumor xenografts with genetically retargeted human T cells containingCD28 and CD137 domains. Proc Natl Acad Sci USA 106:3360-5, 2009; Song DG, Ye Q, Poussin M, et al: CD27 costimulation augments the survival andantitumor activity of redirected human T cells in vivo. Blood119:696-706, 2012), show improved survival and in vivo expansionfollowing adoptive transfer, and more recent clinical trials usinganti-CD19 CAR-modified T cells containing these costimulating moleculeshave shown remarkable efficacy for the treatment of CD19⁺ leukemia.(Kalos M, Levine B L, Porter D L, et al: T cells with chimeric antigenreceptors have potent antitumor effects and can establish memory inpatients with advanced leukemia. Sci Transl Med 3:95ra73, 2011; Porter DL, Levine B L, Kalos M, et al: Chimeric antigen receptor-modified Tcells in chronic lymphoid leukemia. N Engl J Med 365:725-33, 2011;Brentjens R J, Davila M L, Riviere I, et al: CD19-targeted T cellsrapidly induce molecular remissions in adults withchemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med5:177ra38, 2013).

While others have explored additional signaling molecules from tumornecrosis factor (TNF)-family proteins, such as OX40 and 4-1BB, called“third generation” CAR T cells, (Finney H M, Akbar A N, Lawson A D:Activation of resting human primary T cells with chimeric receptors:costimulation from CD28, inducible costimulator, CD134, and CD137 inseries with signals from the TCR zeta chain. J Immunol 172:104-13, 2004;Guedan S, Chen X, Madar A, et al: ICOS-based chimeric antigen receptorsprogram bipolar TH17/TH1 cells. Blood, 2014), other molecules whichinduce T cell signaling distinct from the CD3ζ nuclear factor ofactivated T cells (NFAT) pathway may provide necessary costimulation forT cell survival and proliferation, and possibly endow CAR T cells withadditional, valuable functions, not supplied by more conventionalcostimulating molecules. Some second and third-generation CAR T cellshave been implicated in patient deaths, due to cytokine storm and tumorlysis syndrome caused by highly activated T cells.

A novel T cell costimulating molecule, inducible MyD88/CD40 (iMC)(Narayanan P, Lapteva N, Seethammagari M, et al: A composite MyD88/CD40switch synergistically activates mouse and human dendritic cells forenhanced antitumor efficacy. J Clin Invest 121:1524-34, 2011), has beenfound to provide controlled costimulation to human T cells. IMC is apotent, dimerizer drug (AP1903)-inducible molecule comprising thesignaling elements from both the “universal” Toll-like receptor adapter,MyD88, and the TNF family member, CD40. In these studies, retroviraltransduction of T cells with iMC allows AP1903-dependent signaling, butthis costimulating signal alone was not sufficient to drive IL-2production and T cell proliferation. However, complementing iMC with afirst generation CAR (CD3ζ signaling domain only) allowed complete Tcell activation that required both iMC and tumor recognition through theCAR, resulting in IL-2 production, CD25 receptor upregulation and T cellexpansion, and the therapeutic efficacy was controlled by AP1903 invivo. Further, cells comprising iMC, in the absence of dimerizingligand, still maintain a level of basal activity, which, in the presenceof a co-expressed CAR molecule and antigen, for example tumor antigen,recognition, provides T cell activation. Therefore, these studiesindicate that the chimeric MyD88/CD40 (MC) element is a powerfulcostimulatory molecule for T cells receiving CD3ζ activation followingrecognition of tumor antigen via an extracellular CAR domain.

To extend these initial observations using a binary iMC/CAR system, MCwas assessed for its ability to be included as an intracellularsignaling moiety to provide costimulation to CAR-modified T cells inplace of CD28 or 4-1BB, to provide requisite signaling to enhance T cellsurvival and proliferation. Here, it is shown that MC can be stablyincorporated into the cytoplasmic region of a CAR recognizing prostatestem cell antigen (PSCA), CD19 antigen, or Her2/Neu antigen, andsignaling from this costimulatory molecule enhances tumor cell killingas well as T cell survival and proliferation following tumor cellrecognition.

Further, the chimeric costimulating molecule, MyD88/CD40 (MC), in theabsence of a multimeric ligand-binding region is an intracellularsignaling moiety that activates CAR-expressing cells, such as CAR-Tcells, when expressed as a separate polypeptide from the CAR molecule.Transduction of CAR-T cells with a nucleic acid coding for a MyD88/CD40chimeric stimulating molecule activates the CAR-expressing cells. Thiseffect is observed with a cytoplasmic MyD88/CD40 chimeric stimulatingmolecule, lacking a membrane targeting region, and with a chimericstimulating molecule comprising MyD88/CD40 and a membrane targetingregion, such as, for example, a myristoylation region.

Thus provided in some embodiments is a nucleic acid comprising apromoter operably linked to a polynucleotide encoding a chimericstimulating molecule, wherein the chimeric stimulating moleculecomprises a MyD88 polypeptide or a truncated MyD88 polypeptide lackingthe TIR domain; (ii) a CD40 cytoplasmic polypeptide region lacking theCD40 extracellular domain; and (iii) a membrane targeting region. Alsoprovided is a nucleic acid comprising a promoter operably linked to apolynucleotide encoding a cytoplasmic chimeric stimulating molecule,wherein the cytoplasmic chimeric stimulating molecule comprises a MyD88polypeptide or a truncated MyD88 polypeptide lacking the TIR domain; anda CD40 cytoplasmic polypeptide region lacking the CD40 extracellulardomain. The chimeric stimulating molecules of the present applicationare not capable of ligand-induced multimerization or dimerization causedby the binding of ligand directly to the chimeric stimulating molecules,and do not include multimerizing or dimerizing ligand binding sites,such as, for example, FKBP regions. Also provided in some embodiments,is a nucleic acid comprising a promoter operably linked to apolynucleotide encoding a chimeric stimulating molecule, wherein thechimeric stimulating molecule consists essentially of (i) a MyD88polypeptide or a truncated MyD88 polypeptide lacking the TIR domain;(ii) a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain; and (iii) a membrane targeting region. Alsoprovided in some embodiments is a nucleic acid comprising a promoteroperably linked to a polynucleotide encoding a cytoplasmic chimericstimulating molecule, wherein the cytoplasmic chimeric stimulatingmolecule consists essentially of (i) a MyD88 polypeptide or a truncatedMyD88 polypeptide lacking the TIR domain; and (ii) a CD40 cytoplasmicpolypeptide region lacking the CD40 extracellular domain. By “consistsessentially of” in the context of the chimeric stimulating molecule ismeant that the chimeric stimulating molecule may further includeadditional sequences or regions such as, for example, a linker region,that do not modify the functionality of the (i), (ii), or (iii) regions,and do not include a ligand-induced multimerizing or dimerizing region.

In some embodiments, nucleic acids are provided comprising a promoteroperably linked to a first polynucleotide encoding a chimericstimulating molecule, wherein the chimeric stimulating moleculecomprises (i) a MyD88 polypeptide or a truncated MyD88 polypeptidelacking the TIR domain; (ii) a CD40 cytoplasmic polypeptide regionlacking the CD40 extracellular domain; and (iii) a membrane targetingregion; and a second polynucleotide encoding a chimeric antigenreceptor. In some embodiments, nucleic acids are provided comprising apromoter operably linked to a first polynucleotide encoding acytoplasmic chimeric stimulating molecule, wherein the cytoplasmicchimeric stimulating molecule comprises (i) a MyD88 polypeptide or atruncated MyD88 polypeptide lacking the TIR domain; and (ii) a CD40cytoplasmic polypeptide region lacking the CD40 extracellular domain;and a second polynucleotide encoding a chimeric antigen receptor.

In some embodiments, nucleic acids are provided comprising a promoteroperably linked to a first polynucleotide encoding a chimericstimulating molecule, wherein the chimeric stimulating moleculecomprises (i) a MyD88 polypeptide or a truncated MyD88 polypeptidelacking the TIR domain; (ii) a CD40 cytoplasmic polypeptide regionlacking the CD40 extracellular domain; and (iii) a membrane targetingregion; and a second polynucleotide encoding a T cell receptor or a Tcell receptor based-chimeric antigen receptor. In some embodiments,nucleic acids are provided comprising a promoter operably linked to afirst polynucleotide encoding a cytoplasmic chimeric stimulatingmolecule, wherein the cytoplasmic chimeric stimulating moleculecomprises (i) a MyD88 polypeptide or a truncated MyD88 polypeptidelacking the TIR domain; and (ii) a CD40 cytoplasmic polypeptide regionlacking the CD40 extracellular domain; and a second polynucleotideencoding a T cell receptor or a T cell receptor based chimeric antigenreceptor.

In some embodiments, nucleic acids are provided comprising a promoteroperably linked to a first polynucleotide encoding a chimericstimulating molecule, wherein the chimeric stimulating moleculecomprises (i) a MyD88 polypeptide or a truncated MyD88 polypeptidelacking the TIR domain; (ii) a CD40 cytoplasmic polypeptide regionlacking the CD40 extracellular domain; and (iii) a membrane targetingregion; and a second polynucleotide encoding a chimeric Caspase-9polypeptide comprising a multimeric ligand binding region and aCaspase-9 polypeptide. In some embodiments, nucleic acids are providedcomprising a promoter operably linked to a first polynucleotide encodinga cytoplasmic chimeric stimulating molecule, wherein the cytoplasmicchimeric stimulating molecule comprises (i) a MyD88 polypeptide or atruncated MyD88 polypeptide lacking the TIR domain; and (ii) a CD40cytoplasmic polypeptide region lacking the CD40 extracellular domain;and a second polynucleotide encoding a chimeric Caspase-9 polypeptidecomprising a multimeric ligand binding region and a Caspase-9polypeptide. In some embodiments, nucleic acids are provided comprisinga promoter operably linked to a first polynucleotide encoding a chimericstimulating molecule, wherein the chimeric stimulating moleculecomprises (i) a MyD88 polypeptide or a truncated MyD88 polypeptidelacking the TIR domain; (ii) a CD40 cytoplasmic polypeptide regionlacking the CD40 extracellular domain; and (iii) a membrane targetingregion; a second polynucleotide encoding a chimeric antigen receptor, aT cell receptor, or a T cell receptor based chimeric antigen receptor;and a third polynucleotide encoding a chimeric Caspase-9 polypeptidecomprising a multimeric ligand binding region and a Caspase-9polypeptide.

In some embodiments, nucleic acids are provided comprising a promoteroperably linked to a first polynucleotide encoding a cytoplasmicchimeric stimulating molecule, wherein the cytoplasmic chimericstimulating molecule comprises (i) a MyD88 polypeptide or a truncatedMyD88 polypeptide lacking the TIR domain; and (ii) a CD40 cytoplasmicpolypeptide region lacking the CD40 extracellular domain; and a secondpolynucleotide encoding a chimeric antigen receptor, a T cell receptor,or a T cell receptor based chimeric antigen receptor; and a thirdpolynucleotide encoding a chimeric Caspase-9 polypeptide comprising amultimeric ligand binding region and a Caspase-9 polypeptide.

In certain embodiments, the nucleic acid encodes a chimeric stimulatingmolecule that does not include a membrane targeting region. In certainembodiments, the nucleic acid further comprises a second promoteroperably linked to the second polynucleotide. In certain embodiments,the nucleic acid further comprises a second promoter operably linked tothe second polynucleotide and a third promoter operably linked to thethird polynucleotide. In other embodiments, one promoter is operablylinked to both the first and second polynucleotides, or is operablylinked to the first, second, and third polynucleotides.

In some embodiments, the nucleic acid further comprises a linkerpolynucleotide encoding a linker polypeptide between the first andsecond polynucleotides, wherein the linker polypeptide separates thetranslation products of the first and second polynucleotides during orafter translation. In other embodiments, the nucleic acid furthercomprises polynucleotides encoding linker polypeptides between the threepolynucleotides, wherein the three polynucleotides comprise the first,second, and third polynucleotides, wherein the linker polypeptidesseparate the translation products of the three polynucleotides during orafter translation. In some embodiments, the linker polypeptide is a 2Apolypeptide.

In some embodiments of the present application, a nucleic acid isprovided, comprising a promoter operably linked to a polynucleotideencoding a chimeric antigen receptor, wherein the chimeric antigenreceptor comprises (i) a transmembrane region; (ii) a MyD88 polypeptideor a truncated MyD88 polypeptide lacking a TIR domain; (iii) a CD40cytoplasmic polypeptide region lacking a CD40 extracellular domain; (iv)a T cell activation molecule; and (v) an antigen recognition moiety. Insome embodiments, the chimeric antigen receptor further comprises astalk polypeptide. In some embodiments, a nucleic acid is provided,comprising a promoter operably linked to a first polynucleotide encodinga chimeric antigen receptor, wherein the chimeric antigen receptorcomprises (i) a transmembrane region; (ii) a MyD88 polypeptide or atruncated MyD88 polypeptide lacking a TIR domain; (iii) a CD40cytoplasmic polypeptide region lacking a CD40 extracellular domain; (iv)a T cell activation molecule; and (v) an antigen recognition moiety; anda second polynucleotide encoding a chimeric Caspase-9 polypeptidecomprising a multimeric ligand binding region and a Caspase-9polypeptide. In some embodiments, one promoter is operably linked toboth the first and second polynucleotides. In some embodiments, thenucleic acid further comprises a linker polynucleotide encoding a linkerpolypeptide between the first and second polynucleotides, wherein thelinker polypeptide separates the translation products of the first andsecond polynucleotides during or after translation. In some embodiments,the linker polypeptide is a 2A polypeptide. In some embodiments, thenucleic acid further comprises a second promoter operably linked to thesecond polynucleotide.

In certain embodiments, the membrane targeting region is selected fromthe group consisting of a myristoylation region, palmitoylation region,prenylation region, and transmembrane sequences of receptors. In certainembodiments, the membrane targeting region is a myristoylation region.

In certain embodiments, the truncated MyD88 polypeptide has the aminoacid sequence of SEQ ID NO: 147, or a functional fragment thereof. INcertain embodiments, the MyD88 polypeptide has the amino acid sequenceof SEQ ID NO: 282, or a functional fragment thereof. In certainembodiments, the cytoplasmic CD40 polypeptide has the amino acidsequence of SEQ ID NO: 149, or a functional fragment thereof.

In some embodiments, the nucleic acid is contained within a viralvector. In some embodiments, the viral vector is selected from the groupconsisting of retroviral vectors, murine leukemia virus vectors, SFGvectors, adenoviral vectors, lentiviral vectors, adeno-associated virus(AAV) vectors, Herpes virus vectors, and Vaccinia virus vectors. In someembodiments, the nucleic acid is contained within a plasmid.

Also provided are chimeric stimulating molecule polypeptides encoded bythe nucleic acids of the present application. Thus in some embodiments,chimeric stimulating molecule polypeptides are provided comprising (i) aMyD88 polypeptide or a truncated MyD88 polypeptide lacking the TIRdomain; (ii) a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain; and (iii) a membrane targeting region. Thepolypeptides may be associated with or bound to a membrane in someembodiments. In other embodiments, the polypeptides may be isolated. Inother embodiments, the polypeptides may comprise a membrane targetingregion but not be associated with a membrane. In some embodiments,chimeric stimulating molecule polypeptides are provided comprising aMyD88 polypeptide or a truncated MyD88 polypeptide lacking the TIRdomain; and a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain. The chimeric stimulating molecule polypeptides maybe isolated, or may, in some embodiments, be present in the cytoplasm ofa cell.

Also provided in some embodiments is a chimeric antigen receptorcomprising a MyD88 polypeptide or a truncated MyD88 polypeptide lackinga TIR domain and a CD40 cytoplasmic polypeptide region lacking a CD40extracellular domain encoded by a nucleic acid of the presentapplication. The chimeric antigen receptor may be isolated in someembodiments. In other embodiments, the chimeric antigen receptor may beassociated with or bound to a membrane.

Also provided in some embodiments is a modified cell transfected ortransduced with a nucleic acid encoding a chimeric stimulating moleculeof the present application. In some embodiments, the chimericstimulating molecule is constitutively expressed. In some embodiments,the chimeric stimulating molecule is constitutively active. In someembodiments, the nucleic acid further comprises a polynucleotideencoding a chimeric antigen receptor.

Also provided in some embodiments is a modified cell transfected ortransduced with a nucleic acid encoding a chimeric stimulating moleculeof the present application wherein the nucleic acid does not encode achimeric antigen receptor; and the modified cell further comprises anucleic acid comprising a polynucleotide encoding a chimeric antigenreceptor.

Also provided in some embodiments, is a modified cell transfected ortransduced with a nucleic acid encoding a chimeric stimulating moleculeof the present application wherein the nucleic acid does not encode a Tcell receptor or a T cell receptor based chimeric antigen receptor; andthe modified cell further comprises a nucleic acid comprising apolynucleotide encoding a T cell receptor or a T cell receptorbased-chimeric antigen receptor.

In some embodiments, the modified cells of the present applicationfurther comprise a nucleic acid comprising a polynucleotide encoding achimeric Caspase-9 polypeptide, wherein the chimeric Caspase-9polypeptide comprises a multimeric ligand binding region and a Caspase-9polypeptide.

In some embodiments, the modified cell is a T cell, tumor infiltratinglymphocyte, NK-T cell, TCR-expressing cell, or NK cell. In someembodiments, the cell is a T cell. In some embodiments, the cell isobtained or prepared from bone marrow. In some embodiments, the cell isobtained or prepared from umbilical cord blood. In some embodiments, thecell is obtained or prepared from peripheral blood. In some embodiments,the cell is obtained or prepared from peripheral blood mononuclearcells. In some embodiments, the cell is a human cell. In someembodiments, the cell is transfected or transduced by the nucleic acidvector using a method selected from the group consisting ofelectroporation, sonoporation, biolistics (e.g., Gene Gun withAu-particles), lipid transfection, polymer transfection, nanoparticles,or polyplexes.

In some embodiments, the chimeric Caspase-9 polypeptide comprises aCaspase-9 polypeptide that lacks the CARD domain. In some embodiments,the Caspase-9 polypeptide comprises the amino acid sequence of SEQ IDNO: 153. In some embodiments, the Caspase-9 polypeptide consistsessentially of the amino acid sequence of SEQ ID NO: 153. In certainembodiments, the Caspase-9 polypeptide comprises the amino acid sequenceof SEQ ID NO: 153, and further comprises an amino acid substitutionselected from the group consisting of the caspase variants in Table 1.In certain embodiments, the Caspase-9 polypeptide consists essentiallyof the amino acid sequence of SEQ ID NO: 153, and further comprises anamino acid substitution selected from the group consisting of thecaspase variants in Table 1. In certain embodiments, the Caspase-9polypeptide has a substituted amino acid residue of N405Q. In certainembodiments, the Caspase-9 polypeptide consists essentially of the aminoacid sequence of SEQ ID NO: 153, and further comprises a substitutedamino acid residue of N405Q.

In some embodiments, the multimeric ligand binding domain of thechimeric Caspase-9 polypeptide is selected from the group consisting ofFKBP, cyclophilin receptor, steroid receptor, tetracycline receptor,heavy chain antibody subunit, light chain antibody subunit, single chainantibodies comprised of heavy and light chain variable regions in tandemseparated by a flexible linker domain, and mutated sequences thereof. Insome embodiments, the ligand binding region is an FKBP12 region. In someembodiments, the FKBP12 region is an FKBP12v36 region. In someembodiments, the FKBP region is Fv′Fvls. In some embodiments, the ligandis an FK506 dimer or a dimeric FK506 analog ligand. In some embodiments,the ligand is AP1903 (rimiducid) or AP20187.

The nucleic acids of the present application may comprisepolynucleotides coding for chimeric antigen receptors in someembodiments. In some embodiments, chimeric antigen receptors areexpressed in the modified cells that comprise the nucleic acids of thepresent application. In other embodiments, chimeric antigen receptorsare provided that comprise MyD88 or truncated MyD88 polypeptides and aCD40 cytoplasmic region polypeptide. These chimeric antigen receptors ofthe present application may comprise, in some embodiments, (i) atransmembrane region; (ii) a T cell activation molecule; and (iii) anantigen recognition moiety. In some embodiments, the chimeric antigenreceptor further comprises a co-stimulatory molecule selected from thegroup consisting of CD28, OX40, and 4-1BB. In some embodiments, the Tcell activation molecule is selected from the group consisting of anITAM-containing, Signal 1 conferring molecule, a CD3ζ polypeptide, andan Fc epsilon receptor gamma (FcεR1γ) subunit polypeptide. In someembodiments, the antigen recognition moiety binds to an antigen on atumor cell. In some embodiments, the antigen recognition moiety binds toan antigen on a cell involved in a hyperproliferative disease or to aviral or bacterial antigen. In some embodiments, the antigen recognitionmoiety binds to an antigen selected from the group consisting of PSMA,PSCA, MUC1, CD19, ROR1, Mesothelin, GD2, CD123, MUC16, Her2/Neu, CD20,CD30, PRAME, NY-ESO-1, and EGFRvIII. In some embodiments, the antigenrecognition moiety binds to an antigen selected from the groupconsisting of PSMA, PSCA, MUC1, CD19, ROR1, Mesothelin, GD2, CD123,MUC16, and Her2/Neu. In some embodiments, the antigen recognition moietybinds to PSMA. In some embodiments, the antigen recognition moiety bindsto CD19. In some embodiments, the antigen recognition moiety binds toHer2/Neu.

In some embodiments, the antigen recognition moiety is a single chainvariable fragment. In some embodiments, the transmembrane region is aCD28 transmembrane region or a CD8 transmembrane region. In someembodiments, the chimeric antigen receptor further comprises a CD8 stalkregion.

Methods are provided in some embodiments for stimulating a Tcell-mediated immune response in a subject, comprising administering aneffective amount of modified cells of the present application to thesubject. In some embodiments, the T cell-mediated immune response isdirected against a target cell. In some embodiments, the modified cellcomprises a chimeric antigen receptor, a T cell receptor, or a T cellreceptor based chimeric antigen receptor that binds to an antigen on atarget cell. In some embodiments, the target cell is a tumor cell. Insome embodiments, the number or concentration of target cells in thesubject is reduced following administration of the modified cells.

In some embodiments, the method further comprises measuring the numberor concentration of target cells in a first sample obtained from thesubject before administering the modified cell, measuring the numberconcentration of target cells in a second sample obtained from thesubject after administration of the modified cell, and determining anincrease or decrease of the number or concentration of target cells inthe second sample compared to the number or concentration of targetcells in the first sample. In some embodiments, the concentration oftarget cells in the second sample is decreased compared to theconcentration of target cells in the first sample. In other embodiments,the concentration of target cells in the second sample is increasedcompared to the concentration or target cells in the first sample. Insome embodiments, an additional dose of modified cells is administeredto the subject.

Also provided are methods for providing anti-tumor immunity to asubject, comprising administering to the subject an effective amount ofa modified cell of the present application. Also provided are methodsfor treating a subject having a disease or condition associated with anelevated expression of a target antigen, comprising administering to thesubject an effective amount of a modified cell of the presentapplication. In some embodiments, the target antigen is a tumor antigen.

Also provided in some embodiments are methods for reducing the size of atumor in a subject, comprising administering a modified cell of thepresent application to the subject, wherein the cell comprises achimeric antigen receptor, a T cell receptor, or a T cell receptor basedchimeric antigen receptor comprising an antigen recognition moiety bindsto an antigen on the tumor.

In some embodiments, the subject has been diagnosed as having a tumor.In some embodiments, the subject has cancer. In some embodiments, thesubject has a solid tumor or leukemia. In some embodiments, the modifiedcell is a tumor infiltrating lymphocyte or a T cell. In someembodiments, the modified cell is delivered to a tumor bed. In someembodiments, the cancer is present in the blood or bone marrow of thesubject. In some embodiments the subject has a blood or bone marrowdisease. In some embodiments, the subject has been diagnosed with anycondition or condition that can be alleviated by stem celltransplantation. In some embodiments, the subject has been diagnosedwith sickle cell anemia or metachromatic leukodystrophy. In someembodiments, the subject has been diagnosed with a condition selectedfrom the group consisting of a primary immune deficiency condition,hemophagocytosis lymphohistiocytosis (HLH) or other hemophagocyticcondition, an inherited marrow failure condition, a hemoglobinopathy, ametabolic condition, and an osteoclast condition. In some embodiments,the subject has been diagnosed with a disease or condition selected fromthe group consisting of Severe Combined Immune Deficiency (SCID),Combined Immune Deficiency (CID), Congenital T-cell Defect/Deficiency,Common Variable Immune Deficiency (CVID), Chronic Granulomatous Disease,IPEX (Immune deficiency, polyendocrinopathy, enteropathy, X-linked) orIPEX-like, Wiskott-Aldrich Syndrome, CD40 Ligand Deficiency, LeukocyteAdhesion Deficiency, DOCA 8 Deficiency, IL-10 Deficiency/IL-10 ReceptorDeficiency, GATA 2 deficiency, X-linked lymphoproliferative disease(XLP), Cartilage Hair Hypoplasia, Shwachman Diamond Syndrome, DiamondBlackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia, CongenitalNeutropenia, Sickle Cell Disease, Thalassemia, Mucopolysaccharidosis,Sphingolipidoses, and Osteopetrosis.

In some embodiments, the subject has been diagnosed with an infection ofviral etiology selected from the group consisting HIV, influenza,Herpes, viral hepatitis, Epstein Bar, polio, viral encephalitis,measles, chicken pox, Cytomegalovirus (CMV), adenovirus (ADV), HHV-6(human herpesvirus 6, I), and Papilloma virus, or has been diagnosedwith an infection of bacterial etiology selected from the groupconsisting of pneumonia, tuberculosis, and syphilis, or has beendiagnosed with an infection of parasitic etiology selected from thegroup consisting of malaria, trypanosomiasis, leishmaniasis,trichomoniasis, and amoebiasis.

Also provided are methods of the present application further comprisingdetermining whether an additional dose of the modified cell should beadministered to the subject. In some embodiments, the methods furthercomprise administering an additional dose of the modified cell to thesubject, wherein the disease or condition symptoms remain or aredetected following a reduction in symptoms. In some embodiments, themethods further comprise identifying the presence, absence or stage of acondition or disease in a subject; and transmitting an indication toadminister modified cell of the present application, maintain asubsequent dosage of the modified cell, or adjust a subsequent dosage ofthe modified cell administered to the patient based on the presence,absence or stage of the condition or disease identified in the subject.

In some embodiments, the methods of the present application compriseadministering a modified cell to a subject that comprises a chimericCaspase-9 polypeptide comprising a multimeric ligand binding region anda Caspase-9 polypeptide. In some embodiments the methods furthercomprise administering a multimeric ligand that binds to the multimericligand binding region to the subject following administration of themodified cells to the subject. In some embodiments, after administrationof the multimeric ligand, the number of modified cells comprising thechimeric Caspase-9 polypeptide is reduced. In some embodiments, thenumber of modified cells comprising the chimeric Caspase-9 polypeptideis reduced by 50, 60, 70, 80, 90, 95, or 99% following administration ofthe multimeric ligand to the subject. In some embodiments, the methodscomprise determining that the subject is experiencing a negative symptomfollowing administration of the modified cells to the subject, andadministering the ligand to reduce or alleviate the negative symptom. Insome embodiments, the ligand is AP1903 or AP20187. In some embodiments,the modified cells are autologous T cells. In some embodiments, themodified cells are allogeneic T cells.

In some embodiments, the modified cells of the present application aretransfected or transduced in vivo. In some embodiments, the modifiedcells are transfected or transduced ex vivo.

Also provided in certain embodiments are methods for expressing achimeric stimulating molecule or a chimeric antigen receptor comprisinga MyD88 polypeptide and a CD40 cytoplasmic polypeptide in a cell,comprising contacting a nucleic acid of the present application with acell under conditions in which the nucleic acid is incorporated into thecell, whereby the cell expresses the chimeric stimulating molecule orthe chimeric antigen receptor from the incorporated nucleic acid. Insome embodiments, the nucleic acid is contacted with the cell ex vivo.In some embodiments, the nucleic acid is contacted with the cell invivo.

In some embodiments, the methods further comprise administering achemotherapeutic. In certain embodiments, the chemotherapeutic selectedis a lymphodepleting chemotherapeutic. In some embodiments, the modifiedcells, or the nucleic acid, and the chemotherapeutic agent areadministered in an amount effective to treat the disease or condition inthe subject. In some embodiments, the chemotherapeutic agent is selectedfrom the group consisting of carboplatin, estramustine phosphate(Emcyt), and thalidomide. In some embodiments, the chemotherapeuticagent is a taxane. The taxane may be, for example, selected from thegroup consisting of docetaxel (Taxotere), paclitaxel, and cabazitaxel.In some embodiments, the taxane is docetaxel. In some embodiments, thechemotherapeutic agent is administered at the same time or within oneweek after the administration of the modified cell or nucleic acid. Inother embodiments, the chemotherapeutic agent is administered from 1 to4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 monthsafter the administration of the modified cell or nucleic acid. In someembodiments, the chemotherapeutic agent is administered at least 1 monthbefore administering the cell or nucleic acid.

In some embodiments, the methods further comprise administering two ormore chemotherapeutic agents. In some embodiments, the chemotherapeuticagents are selected from the group consisting of carboplatin,Estramustine phosphate, and thalidomide. In some embodiments, at leastone chemotherapeutic agent is a taxane. The taxane may be, for example,selected from the group consisting of docetaxel, paclitaxel, andcabazitaxel. In some embodiments, the taxane is docetaxel. In someembodiments, the chemotherapeutic agents are administered at the sametime or within one week after the administration of the modified cell ornucleic acid. In other embodiments, the chemotherapeutic agents areadministered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1week to 123 months after the administration of the cell or nucleic acid.In other embodiments, the methods further comprise administering thechemotherapeutic agents from 1 to 4 weeks or from 1 week to 1 month, 1week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9months, or 1 week to 123 months before the administration of the cell ornucleic acid.

In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human.

This application incorporates by reference U.S. patent application Ser.No. 14/210,034, titled METHODS FOR CONTROLLING T CELL PROLIFERATION,filed Mar. 13, 2014; U.S. patent application Ser. No. 14/622,018, filedFeb. 13, 2015, titled METHODS FOR ACTIVATING T CELLS USING AN INDUCIBLECHIMERIC POLYPEPTIDE; U.S. patent application Ser. No. 13/112,739, filedMay 20, 2011, titled METHODS FOR INDUCING SELECTIVE APOPTOSIS; U.S.patent application Ser. No. 13/792,135, filed Mar. 10, 2013, titledMODIFIED CASPASE POLYPEPTIDES AND USES THEREOF; AND U.S. patentapplication Ser. No. 14/296,404, filed Jun. 4, 2014, titled METHODS FORINDUCING PARTIAL APOPTOSIS USING CASPASE POLYPEPTIDES; which are allhereby incorporated by reference herein in their entirety.

Also incorporated by reference in their entirety are U.S. Pat. No.7,404,950, issued Jul. 29, 2008, to Spencer, D. et al.; U.S. Pat. No.8,691,210, issued Apr. 8 2004 to Spencer, et al.; U.S. patentapplication Ser. No. 12/532,196 by Spencer et al., filed Sep. 21, 2009;PCT application PCT/US2009/057738 to Spencer et al., published on Apr.24, 2008 as WO2010/033949; U.S. patent application Ser. No. 13/087,329by Slawin et al., filed Apr. 14, 2011; PCT applicationPCT/US2011/032572, published on Oct. 20, 2011 as WO2011/130566; U.S.patent application Ser. No. 14/210,324 by Spencer et al., filed Mar. 13,2014; PCT application number PCT/US2014/026734 by Spencer et al.,published as WO2014/251960 on Feb. 5, 2015; U.S. application Ser. No.14/622,018, by Foster et al., filed Feb. 13, 2015; PCT applicationnumber PCT/US2015/015829 by Foster et al., published as WO2015/123527 onAug. 20, 2015

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 is a schematic of a partial view of a cell membrane of a celltransduced or transfected with a chimeric antigen receptor and anexample of a chimeric stimulating molecule comprising CD28 and 4-1BBpolypeptides.

FIG. 2 is a schematic of a partial view of a cell membrane of a celltransduced or transfected with a chimeric antigen receptor and anexample of a chimeric signaling molecule comprising an induciblechimeric CD40/MyD88 polypeptide.

FIG. 3A-FIG. 3D provide examples of inducible chimeric stimulatingmolecules comprising CD40 and MyD88 polypeptides. FIG. 3A provides agraphic illustration of the general polypeptide elements of theinducible chimeric stimulating molecules. FIG. 3B provides flowcytometry results of CD19 marker detection in T cells that express thechimeric stimulating molecules. FIG. 3C is a bar graph of IFN γproduction in T cells that express the chimeric stimulating molecules.FIG. 3D is a bar graph of IL-6 production in T cells that express thechimeric stimulating molecules.

FIG. 4A-FIG. 4D provide examples of chimeric antigen receptors. FIG. 4Aprovides a graphic illustration of chimeric antigen receptors comprisingMyD88 and CD40 polypeptides in the general context of a cell membrane.FIG. 4B is a bar graph of CAR expression in the transduced cells. FIG.4C provides flow cytometry results showing CAR expression in thetransduced cells. FIG. 4D is a bar graph showing specific lysis of PSCA⁺tumor cells by the CAR-expressing T cells.

FIG. 5A-FIG. 5C provide examples of endogenous costimulation of the CARmolecule by MyD88 and CD40 polypeptides. FIG. 5A provides flow cytometryresults measuring the killing of Capan-1-GFP cells by cells expressingthe chimeric antigen receptor. FIG. 5B is a bar graph of the killing ofCapan-1-GFP cells at a 1:1 ratio of CAR T cells to tumor cells. FIG. 5Cis a bar graph of the killing of Capan-1-GFP cells at a 1:10 ratio ofCART cells to tumor cells.

FIG. 6A-FIG. 6D provide results of cell viability and proliferation ofcells that express the MyD88/CD40 chimeric antigen receptor. FIG. 6A isa graph of T cell viability. FIG. 6B is a graph of cell proliferation.FIG. 6C is a graph of IL-2 production. FIG. 6D is a graph of IL-6production.

FIG. 7 is a plasmid map coding for a chimeric antigen receptorco-expressed with an inducible caspase molecule.

FIG. 8 is a plasmid map coding for a chimeric stimulating molecule and aCD19 polypeptide marker.

FIG. 9A-FIG. 9D: FIG. 9A is a schematic of a chimeric antigen receptorcomprising MyD88 and CD40 polypeptides. FIG. 9B is a bar graph of anIL-2 assay of cells transfected with a plasmid encoding a chimericantigen receptor comprising MyD88 and CD40 polypeptides. FIG. 9C is aschematic of a MyD88/CD40 costimulating molecule co-expressed with afirst generation chimeric antigen receptor. FIG. 9D is a bar graph of anIL-2 assay of cells that express both a MyD88 costimulating molecule anda chimeric antigen receptor.

FIG. 10 is a plasmid map coding for a MyD88/CD40 chimeric antigenreceptor co-expressed with an inducible caspase-9 polypeptide.

FIG. 11 is a schematic for a transgene of the MyD88/CD40 CARco-expressed with an inducible caspase-9 polypeptide.

FIG. 12A-FIG. 12C provide results of experiments comparing a firstgeneration CAR (CD19ζ) a CAR including the CD3ζ and 4-1 BB polypeptides,and a CAR including the CD3ζ and MyD88/CD40 polypeptides. FIG. 12Aprovides flow cytometry results of transduction efficiency. FIG. 12B isa graph of transduction efficiency. FIG. 12C is a graph of CARexpression level.

FIG. 13A-FIG. 13B provide results of experiments comparing a firstgeneration CAR (CD19ζ) a CAR including the CD3ζ and 4-1 BB polypeptides,and a CAR including the CD3ζ and MyD88/CD40 polypeptides for IL-2production and IL-6 production following co-culture with CD19⁺ DaudiBurkitt's lymphoma cells. NT denotes non-transformed cells. FIG. 13A isa graph of IL-2 production. FIG. 13B is a graph of IL-6 production.

FIG. 14A-FIG. 14B provide results of experiments comparing a firstgeneration CAR (CD19ζ) a CAR including the CD3ζ and 4-1 BB polypeptides,and a CAR including the CD3ζ and MyD88/CD40 polypeptides for IL-2production and IL-6 production following co-culture with CD19⁺ RajiBurkitt's lymphoma cells. NT denotes non-transformed cells. FIG. 14A isa graph of IL-2 production. FIG. 14B is a graph of IL-6 production.

FIG. 15A-FIG. 15B provide results of tumor cell killing experimentscomparing a first generation CAR (CD19ζ) a CAR including the CD3ζ and4-1 BB polypeptides, and a CAR including the CD3ζ and MyD88/CD40polypeptides following 6 days of co-culture with FIG. 15A CD19⁺ DaudiBurkitt's lymphoma cells and FIG. 15B CD19⁺ Raji Burkitt's lymphomacells. NT denotes non-transformed cells.

FIG. 16A-FIG. 16B provide schematics of a first generation-type chimericantigen receptor (FIG. 16A), and a MyD88/CD40 chimeric antigen receptor(FIG. 16B).

FIG. 17 is a plasmid map coding for a MyD88/CD40 chimeric antigenreceptor comprising an antigen recognition moiety that recognizesHer2/Neu antigen.

FIG. 18A-FIG. 18B provide examples of a Her2/New targeted chimericantigen receptor. FIG. 18A provides a schematic of a Her2/Neu targetedCAR with and without the MC signaling domain; FIG. 18B provides sortingmap results of the transduction of T cells and subsequent measurement ofCAR expressed on the T cells using the CD34 epitope as a marker by flowcytometry.

FIG. 19A-FIG. 19B provide graphs of T cell viability and T cell countfollowing co-culturing of CAR T cells wherein the antigen recognitionmoiety of the chimeric antigen receptor recognizes the Her2/Neu antigen,with the Her2⁺ breast cancer cell line MCF-7 for 7 days. These graphsshow the viability of T cells after coculture and the total T cellnumber, indicating that CARs containing the MC molecule have bettersurvival and enhanced proliferation. FIG. 19A is a graph of T cellviability. FIG. 19B is a graph of T cell count.

FIG. 20A-FIG. 20B provide data showing the results of a co-culture assaywith CAR-modified T cells, where the antigen recognition moiety of thechimeric antigen receptor recognizes the Her2/Neu antigen. FIG. 20A Tcells were cultured with Her2⁺ MCF-7-GFP tumor cells and tumor killingwas assessed by measuring the percentage of GFP cells remaining in theculture after 7 days. FIG. 20B is a graph of cumulative data from twoseparate experiments.

FIG. 21A-FIG. 21E provide data showing that the MyD88/CD40 signalingdomain enhances T cell activity in a CD19 CAR. FIG. 21A provides flowcytometry data of cytotoxicity of non-transduced (NT), CD19.ζ andCD19.MC. CAR modified T cells against CD19+ Raji tumor cells a 5:1effector to target (E:T) ratio. FIG. 21B is a line graph of cytotoxicitydilution of CAR constructs against Raji tumor cells. FIG. 21C is a bargraph of IL-2 production after 48 hours of coculture with Raji tumorcells. FIG. 21D is a bar graph of IL-6 production after 48 hours ofcoculture with Raji tumor cells. FIG. 21E is a bar graph of cell numberof CAR modified T cells after 4 days of culture with Raji tumor cells.

FIG. 22 is a plasmid map coding for a MyD88/CD40 chimeric antigenreceptor comprising an antigen recognition moiety that recognizes CD19,and an inducible caspase-9 polypeptide.

FIG. 23 is a plasmid map coding for a MyD88/CD40 chimeric antigenreceptor comprising an antigen recognition moiety that recognizes PSCA.

FIG. 24 is a plasmid map coding for a MyD88/CD40 chimeric antigenreceptor comprising and antigen recognition moiety that recognizes CD19.

FIG. 25 is a plasmid map coding for a MyD88/CD40 chimeric stimulatingmolecule that is co-expressed with a chimeric antigen receptor.

FIG. 26A-FIG. 26D are schematics showing the CAR vector design discussedin Example 17. Four vectors were constructed bearing the iCaspase-9suicide gene (iCasp9). FIG. 26A represents a standard 1st generation CARmolecule. FIG. 26B depicts a CAR that includes the CD28 endodomain. FIG.26C: MC is expressed within the CAR molecule or as a constitutivelyexpressed protein using a second 2A (FIG. 26D).

FIG. 27A and FIG. 27B provide graphs of CD3⁺CD34⁺% (FIG. 27A) and MFI(FIG. 27B) in T cells transduced with CAR molecules with different MCformats. T cells were transduced with each of the four vectors andcompared to non-transduced T cells (NT) for CAR transduction efficiency(FIG. 27A) and CAR expression level (FIG. 27B) using mean fluorescenceintensity (MFI) following CD3 and CD34 antibody labeling.

FIG. 28A and FIG. 28B provide graphs of IL-2 (FIG. 28A) and IL-6 (FIG.28B) production following CD19\+ Raji coculture. T cells were transducedwith each of the 4 vectors and compared to non-transduced T cells forcytokine production after a 1:1 T cell to tumor cell ratio cocultureassay using Raji tumor cells. Supernatants were harvested at 48 hoursand measured for IL-2 and IL-6 by ELISA.

FIG. 29A and FIG. 29B are graphs of CD3⁺% (FIG. 29A) and Raji-GFP %(FIG. 29B), assaying antitumor activity of different MC formats. T cellswere transduced with each of the 4 vectors and compared tonon-transduced T cells for tumor killing following coculture at a 1:1 Tcell to tumor cell ratio using Raji-GFP tumor cells. After 14 days, theculture was harvested and analyzed by flow cytometry for CD3+ T cellsand GFP+ tumor cells.

FIG. 30A and FIG. 30B are graphs of T cell number (FIG. 30A) andRaji-GFP (FIG. 30B) following MC costimulation of CD19-targeted CAR Tcells. T cells were transduced with each of the 4 vectors and comparedto non-transduced T cells for tumor killing following coculture at a 1:1T cell to tumor cell ratio using Raji-GFP tumor cells. After 14 days,the culture was harvested and analyzed by flow cytometry for CD3+ Tcells and GFP+ tumor cells and the cell numbers calculated base offtotal cell numbers.

FIG. 31 is a FACs plot of expression of the CAR molecule with differentMC formats using a Her2-targeted CAR. T cells were transduced with fivedifferent vectors targeting Her2 and subsequently analyzed by flowcytometry for CAR expression on days 4 and 8 post-transduction using CD3and CD34 antibodies.

FIG. 32A and FIG. 32B are graphs of CAR transduction percent and MFI forHER2 CAR constructs of CD3⁺CD34⁺% (FIG. 32A) and MFI (FIG. 32B).

FIG. 33A and FIG. 33B are graphs of IL-2 (FIG. 33A) and IL-6 (FIG. 33B)production for HER2 CAR constructs.

FIG. 34A and FIG. 34B provide graphs of antitumor activity of differentMC formats; FIG. 34A: CD3+%, FIG. 34B:SK-BR-3-GFP %. T cells weretransduced with each of the 5 vectors and compared to non-transduced Tcells for tumor killing following coculture at a 1:1 T cell to tumorcell ratio using Her2+SK-BR-3-GFP tumor cells. After 14 days, theculture was harvested and analyzed by flow cytometry for CD3+ T cellsand GFP+ tumor cells.

FIG. 35A and FIG. 35B provide graphs showing MCcostimulation enhances Tcell proliferation of Her2-targeted CAR-T cells. FIG. 35A: T cellnumber, FIG. 35B: SK-BR-3-GFP. T cells were transduced with each of the4 vectors and compared to non-transduced T cells for tumor killingfollowing coculture at a 1:1 T cell to tumor cell ratio usingSK-BR-3-GFP tumor cells. After 14 days, the culture was harvested andanalyzed by flow cytometry for CD3+ T cells and GFP+ tumor cells and thecell numbers calculated base off total cell numbers.

FIG. 36A and FIG. 36B provide graphs showing that constitutivelyexpressed MC using a 2A polypeptide demonstrates enhanced antitumoractivity in vivo. FIG. 36A: Tumor size, FIG. 36B: Weight change %.Immune deficient mice (NSG) were injected s.c. with 1×106SK-BR-3-EGFPluciferase tumor cells in the right flank. After 7 days, thetumors were treated with 2 intratumoral injections on days 7 and 10 with1×107 Her2-specific CAR-modified T cells, or non-transduced (NT). Tumorsize and weight was measured twice weekly for each of the groups.

FIG. 37A-FIG. 37E provide schematics of retrovirus constructs used toexpress chimeric antigen receptors containing single chain variablefragments specific for CD19 and Her2. The constructs also includedpolynucleotides coding for various costimulating molecules such as theCD28 costimulatory domain, or MyD88, CD40, or MyD88/CD40 (MC). Theconstructs also include a polynucleotide sequence coding for a Caspase-9safety switch (iC9). FIG. 37A: iC9-scFv.ζ FIG. 37B: iC9-scFv.28.ζ FIG.37C: iC9-scFv.ζ-CD40. FIG. 37D: iC9-scFv.ζ-MyD88. FIG. 37E:iC9-scFv.ζ-MC.

FIG. 38A-FIG. 38F provide results of MyD88/CD40 (MC) costimulationassays using the constructs of FIG. 37A-FIG. 37E. FIG. 38A provides flowcytometry results showing expression of the CAR/chimeric costimulatingmolecule construct in T cells. FIG. 38B provides a bar graph of IL-2production in T cells expressing the various constructs. FIG. 38Cprovides a bar graph of the number of T cells from a cocultureexperiment with different CAR constructs. FIG. 38D provides a bar graphtumor cell number from a coculture experiment with different CARconstructs. FIG. 38E provides flow cytometry results showing efficacy oftumor cell elimination following coculture with T cells expressing thevarious constructs. FIG. 38F provides a bar graph of IL-2 productionfrom a coculture assay using the various constructs.

FIG. 39A-FIG. 39F provide results of the enhancement of Her2CAR-T cellefficacy in vivo by the MyD88/CD40 chimeric costimulatory molecule usingthe constructs of FIG. 37A-FIG. 37E. FIG. 39A T cells were transducedwith Her2.ζ, Her2.28.ζ or Her2.ζ-MC CARs and injected directly intoluciferase-expressing s.c. Her2+SK-BR-3 tumors engrafted into NSG mice(n=5). Bioluminescence (BLI), as shown in FIG. 39A photos, of tumorcells was measured by IVIS. FIG. 39B is a graph of tumor size, which wasmeasured by calipers and survival. FIG. 39C is a graph of tumor sizecalculated over 75 days. FIG. 39D is a photo showing bioluminescencefollowing subsequent co-transduction of T cells with CAR and luciferaseand injected directly into s.c. Her2⁺ SK-BR-3 tumors engrafted into NSGmice (n=5). FIG. 39E is a graph of CAR-T cell expansion which wascalculated by region-of-interest ROI using IVIS imaging. FIG. 39F is agraph of tumor size, which was calculated by caliper measurements andshows individual mice in each treatment group. *P-value=<0.05.

FIG. 40A-FIG. 40C provide results of the enhancement of CD19 CAR-T cellefficacy in vivo by the MyD88/CD40 chimeric costimulatory molecule usingthe constructs of FIG. 37A-FIG. 37E. FIG. 40A: NSG mice (n=5 per group)were engrafted with Raji-luciferase tumor cells and then treated withnon-transduced (NT) or iC9-CD19.ζ-MC CAR-modified T cells on day 3.Tumor growth was measured by IVIS imaging and calculated by whole-bodyBLI, as shown in photos. FIG. 40B provides a graph of bioluminescentcounts (BLI counts) and FIG. 40C provides a graph of (Kaplan-Meieranalysis from (A)). At objective evidence of sCRS, rimiducid (AP1903)was administered (gray boxes, FIG. 40A; third row middle column, secondrow right column), leading to normalization of cytokines within 24 hrsand complete resolution of clinical sCRS without compromising tumorcontrol.

FIG. 41A-FIG. 41C provide results of assays of the titration ofinducible chimeric Caspase-9 expressing cells, using the constructs ofFIG. 37A-FIG. 37E, with rimiducid. FIG. 41A and FIG. 41B: NSG mice (n=5per group) were engrafted with CD19⁺ Raji lymphoma cells and treatedwith 5×10⁶ iC9-CD19.ζ-MC/luciferase-transduced T cells at day 3. After 6days, mice were treated i.p. with log dilutions of rimiducid (0.00005-5mg/kg). BLI of CAR-T cells was assessed prior to rimiducid treatment andat 24 and 48 hours post-injection. FIG. 41A provides photos showingbioluminescence, and FIG. 41B provides graphs of the results of theassay. FIG. 41C provides graphs of serum cytokine levels, which weremeasured from each group before (black line) and 24 hourspost-administration (gray line) of rimiducid. *P-value=<0.01.

FIG. 42A-FIG. 42E provide the results of efficacy and safety assays ofvarious Her2-chimeric antigen receptor constructs of FIG. 37A-FIG. 37E.FIG. 42A provides a timeline of the assays. FIG. 42B-FIG. 42E providegraphs showing tumor size in mice following administration of control Tcells (FIG. 42B) and transduced T cells. FIG. 42C: Her2.ζ. FIG. 42D:Her2.28.ζ. FIG. 42E: Her2.ζ-MC.

FIG. 43A-FIG. 43C provide the results of efficacy and safety assays ofvarious Her2-chimeric antigen receptor constructs of FIG. 37A-FIG. 37E.FIG. 43A provides a timeline of the assays. FIG. 43B providesphotographs showing bioluminescence in mice following administration ofthe transduced T cells. FIG. 43C is a graph of the bioluminescenceassay.

FIG. 44A-FIG. 44D provide results of assays of the titration ofinducible chimeric Caspase-9 expressing cells, using the CD19-specificCAR constructs of FIG. 37A-FIG. 37E, with rimiducid. FIG. 44A providesphotographs showing bioluminescence in mice following administration ofthe transduced T cells. FIG. 44B is a graph of bioluminescence in micefollowing administration of rimiducid to induce Caspase-9 activity. FIG.44C is a graph of IL-6 secretion, and FIG. 44D is a graph of TNF-alphasecretion following administration of rimiducid.

FIG. 45A-FIG. 45D provide graphs of tumor size in days postadministration of Her2-specific CAR constructs. FIG. 45A: non-transducedT cell control. FIG. 45B: iC9-Her2. FIG. 45C: iC9-Her2.28. FIG. 45D:iC9-Her2.ζ-MC.

FIG. 46A-FIG. 46C provide results of assays of in vivo expansion ofMC-enabled Her2 CAR-T cells. FIG. 46A provides photographs showingbioluminescence in mice following administration of the transduced Tcells. FIG. 46B provides a graph of tumor size in dayspost-administration. FIG. 46C provides a graph of bioluminescence indays post-administration.

FIG. 47A-FIG. 47C provide results of assays of survival of micefollowing administration of MC-enabled Her2 CAR-T cells. FIG. 47Aprovides a graph of tumor size. FIG. 47B provides a graph ofbioluminescence, and FIG. 47C provides a graph of percent survival indays post-injection of T cells.

FIG. 48A-FIG. 48F provide schematic representations of the DNAconstructs used to express MyD88/CD40 chimeric stimulating molecules.FIG. 48A: construct encodes a CD19-specific chimeric antigen receptor, achimeric inducible Caspase-9 polypeptide, and a MyD88/CD40 costimulatorymolecule. FIG. 48B: construct encodes a CD19-specific chimeric antigenreceptor and a chimeric inducible Caspase-9 polypeptide. FIG. 48C:construct encodes a CD19-specific chimeric antigen receptor. FIG. 48D:construct encodes a membrane-targeted MyD88/CD40 costimulatory moleculeand a CD19-specific chimeric antigen receptor. FIG. 48E constructencodes a membrane-targeted MyD88/CD40 costimulatory molecule comprisingan epitope for the QBEND10 antibody directed to CD34 (Q epitope). FIG.48F: construct encodes a membrane-targeted inducible MyD88/CD40costimulatory molecule comprising a multimeric ligand binding site and aCD19-specific chimeric antigen receptor.

FIG. 49 provides a schematic representation of the proteins produced bythe constructs in FIG. 48. Dots represent the plasma membrane with MC in1152 tethered to the membrane with an N-terminal myristate.

FIG. 50A and FIG. 50B are graphs of IL-2 secretion from T cellstransduced with the indicated recombinant retroviruses. FIG. 50A: assayperformed at 24 hours post transduction. FIG. 50B: assay performed at 48hours post transduction.

FIG. 51A and FIG. 51B are graphs of IL-6 secretion from T cellstransduced with the indicated recombinant retroviruses. FIG. 51A: assayperformed at 24 hours post transduction. FIG. 51B: assay performed at 48hours post transduction.

DETAILED DESCRIPTION

Adoptive transfer of T cells genetically engineered to express chimericantigen receptors (CARs) that recognize antigens expressed on tumorcells have begun to show promise in clinical studies. CARs are comprisedof an antigen binding region, for example, a single-chain variablefragment (scFv) derived from an antigen-specific monoclonal antibody anda T cell activation molecule, such as the ζ-chain from the T cellreceptor (CD3).

The basic components of a chimeric antigen receptor (CAR) include thefollowing. The variable heavy (VH) and light (VL) chains for atumor-specific monoclonal antibody are fused in-frame with the CD3ζchain (ζ) from the T cell receptor complex. The VH and VL are generallyconnected together using a flexible glycine-serine linker, and thenattached to the transmembrane domain by a spacer (CH₂CH₃) to extend thescFv away from the cell surface so that it can interact with tumorantigens.

Following transduction, T cells now express the CAR on their surface,and upon contact and ligation with a tumor antigen, signal through theCD3ζ chain inducing cytotoxicity and cellular activation.

Investigators have noted that activation of T cells through CD3ζ issufficient to induce a tumor-specific killing, but is insufficient toinduce T cell proliferation and survival. Early clinical trials using Tcells modified with CARs expressing only the ζ chain showed thatgene-modified T cells exhibited poor survival and proliferation in vivo.These constructs are termed 1st generation CARs.

As co-stimulation through the B7 axis is necessary for complete T cellactivation, investigators added the co-stimulating polypeptide CD28signaling domain to the CAR construct. This region generally containsthe transmembrane region (in place of the CD3ζ version) and the YMNMmotif for binding PI3K and Lck. In vivo comparisons between T cellsexpressing CARs with only or CARs with both ζ and CD28 demonstrated thatCD28 enhanced expansion in vivo, in part due to increased IL-2production following activation. The inclusion of CD28 is called a 2ndgeneration CAR. The most commonly used costimulating molecules includeCD28 and 4-1BB, which, following tumor recognition, can initiate asignaling cascade resulting in NF-κB activation, which promotes both Tcell proliferation and cell survival.

The use of co-stimulating polypeptides 4-1BB or OX40 in CAR design hasfurther improved T cell survival and efficacy. 4-1 BB in particularappears to greatly enhance T cell proliferation and survival. This 3rdgeneration design (with 3 signaling domains) has been used in PSMA CARs(Zhong X S, et al., Mol Ther. 2010 February; 18(2):413-20) and in CD19CARs, most notably for the treatment of CLL (Milone, M. C., et al.,(2009) Mol. Ther. 17:1453-1464; Kalos, M., et al., Sci. Transl. Med.(2011) 3:95ra73; Porter, D., et al., (2011) N. Engl. J. Med. 365:725-533). These cells showed impressive function in 3 patients,expanding more than a 1000-fold in vivo, and resulted in sustainedremission in all three patients.

T cell receptor signaling can be induced using a chemical inducer ofdimerization (CID) in combination with a chimeric receptor that includesa multimerization region that binds to the CID, T cells were engineeredto express the CD3ζ chain, which was linked with 1, 2, or 3 FKBPfragments. The cells expressed the chimeric receptor, and demonstratedCID-dependent T cell activation (Spencer, D. M., et al., Science, 1993.262: p. 1019-1024). An inducible MyD88/CD40 (iMC) molecule for theactivation of CAR-modified T cells was tested, and it was found thatactivation of iMC by AP1903 (rimiducid) provides powerful costimulationthat increases T cell survival, proliferation, activation and tumor cellkilling.

An inducible MyD88/CD40 (MC) molecule, when co-expressed in a T cellwith a CAR molecule comprising CD3ζ, was found to provide costimulation.In this assay, the MyD88/CD40 molecule also included a multimerizationregion, and was inducible in the presence of the AP1903 ligand. Theinducible MyD88/CD40 polypeptide was coexpressed with a CD19-bindingchimeric antigen receptor. In the absence of dimerizing ligand, basalactivity was observed, allowing high IL-2 production.

Next, a MyD88/CD40 molecule was assayed to determine whether it couldalso be used to replace CD28 or 4-1BB costimulation in CAR designs. Thefunctionality of MyD88/CD40 as a costimulating molecule to prostate stemcell antigen (PSCA)-targeted CARs was assayed with either CD3ζ (PSCA.ζ)or CD28.CD3ζ (PSCA.28.ζ) endodomains and the data showed thatincorporation of MC promoted T cell survival and proliferation, enhancedtumor killing in co-culture assays against a PSCA⁺ tumor cell line(Capan-1) and improved cytokine production (e.g., IL-2 and IL-6)compared to T cells transduced with only PSCA.ζ. MyD88/CD40 cantherefore be used, for example, as a costimulatory endodomain to enhancethe function of CAR T cells.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having”, “including”, “containing” and “comprising” are interchangeableand one of skill in the art is cognizant that these terms are open endedterms.

The term “allogeneic” as used herein, refers to HLA or MHC loci that areantigenically distinct between the host and donor cells.

Thus, cells or tissue transferred from the same species can beantigenically distinct. Syngeneic mice can differ at one or more loci(congenics) and allogeneic mice can have the same background.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.Exemplary organisms include but are not limited to, Helicobacters,Campylobacters, Clostridia, Corynebacterium diphtheriae, Bordetellapertussis, influenza virus, parainfluenza viruses, respiratory syncytialvirus, Borrelia burgdorferi, Plasmodium, herpes simplex viruses, humanimmunodeficiency virus, papillomavirus, Vibrio cholera, E. coli, measlesvirus, rotavirus, shigella, Salmonella typhi, Neisseria gonorrhea.Therefore, any macromolecules, including virtually all proteins orpeptides, can serve as antigens. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. Any DNA that contains nucleotidesequences or partial nucleotide sequences of a pathogenic genome or agene or a fragment of a gene for a protein that elicits an immuneresponse results in synthesis of an antigen. Furthermore, the presentmethods are not limited to the use of the entire nucleic acid sequenceof a gene or genome. The present compositions and methods include, butare not limited to, the use of partial nucleic acid sequences of morethan one gene or genome and that these nucleic acid sequences arearranged in various combinations to elicit the desired immune response.

The term “antigen-presenting cell” is any of a variety of cells capableof displaying, acquiring, or presenting at least one antigen orantigenic fragment on (or at) its cell surface. In general, the term“cell” can be any cell that accomplishes the goal of aiding theenhancement of an immune response (i.e., from the T-cell or -B-cell armsof the immune system) against an antigen or antigenic composition. Asdiscussed in Kuby, 2000, Immunology, .supp. 4th edition, W.H. Freemanand company, for example, (incorporated herein by reference), and usedherein in certain embodiments, a cell that displays or presents anantigen normally or with a class II major histocompatibility molecule orcomplex to an immune cell is an “antigen-presenting cell.” In certainaspects, a cell (e.g., an APC cell) may be fused with another cell, suchas a recombinant cell or a tumor cell that expresses the desiredantigen. Methods for preparing a fusion of two or more cells arediscussed in, for example, Goding, J. W., Monoclonal Antibodies:Principles and Practice, pp. 65-66, 71-74 (Academic Press, 1986);Campbell, in: Monoclonal Antibody Technology, Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burden & Von Knippenberg,Amsterdam, Elseview, pp. 75-83, 1984; Kohler & Milstein, Nature,256:495-497, 1975; Kohler & Milstein, Eur. J. Immunol., 6:511-519, 1976,Gefter et al., Somatic Cell Genet., 3:231-236, 1977, each incorporatedherein by reference. In some cases, the immune cell to which a celldisplays or presents an antigen to is a CD4⁺TH cell. Additionalmolecules expressed on the APC or other immune cells may aid or improvethe enhancement of an immune response. Secreted or soluble molecules,such as for example, cytokines and adjuvants, may also aid or enhancethe immune response against an antigen. Various examples are discussedherein.

An “antigen recognition moiety” may be any polypeptide or fragmentthereof, such as, for example, an antibody fragment variable domain,either naturally derived, or synthetic, which binds to an antigen.Examples of antigen recognition moieties include, but are not limitedto, polypeptides derived from antibodies, such as, for example, singlechain variable fragments (scFv), Fab, Fab′, F(ab′)₂, and Fv fragments;polypeptides derived from T Cell receptors, such as, for example, TCRvariable domains; secreted factors (e.g., cytokines, growth factors)that can be artificially fused to signaling domains (e.g., “zytokines”),and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds tothe extracellular cognate protein. Combinatorial libraries could also beused to identify peptides binding with high affinity to tumor-associatedtargets. Moreover, “universal” CARs can be made by fusing aviden to thesignaling domains in combination with biotinylated tumor-targetingantibodies (Urbanska (12) Ca Res) or by using Fc gamma receptor/CD16 tobind to IgG-targeted tumors (Kudo K (13) Ca Res).

The term “autologous” means a cell, nucleic acid, protein, polypeptide,or the like derived from the same individual to which it is lateradministered. The modified cells of the present methods may, forexample, be autologous cells, such as, for example, autologous T cells.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma,astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck,breast, pancreatic, prostate, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,sarcoma or bladder.

The terms “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

By “chimeric antigen receptor” or “CAR” is meant, for example, achimeric polypeptide which comprises a polypeptide sequence thatrecognizes a target antigen (an antigen-recognition domain) linked to atransmembrane polypeptide and intracellular domain polypeptide selectedto activate the T cell and provide specific immunity. Theantigen-recognition domain may be a single-chain variable fragment(ScFv), or may, for example, be derived from other molecules such as,for example, a T cell receptor or Pattern Recognition Receptor. Theintracellular domain comprises at least one polypeptide which causesactivation of the T cell, such as, for example, but not limited to, CD3zeta, and, for example, co-stimulatory molecules, for example, but notlimited to, CD28, OX40 and 4-1BB. The term “chimeric antigen receptor”may also refer to chimeric receptors that are not derived fromantibodies, but are chimeric T cell receptors. These chimeric T cellreceptors may comprise a polypeptide sequence that recognizes a targetantigen, where the recognition sequence may be, for example, but notlimited to, the recognition sequence derived from a T cell receptor oran scFv. The intracellular domain polypeptides are those that act toactivate the T cell. Chimeric T cell receptors are discussed in, forexample, Gross, G., and Eshar, Z., FASEB Journal 6:3370-3378 (1992), andZhang, Y., et al., PLOS Pathogens 6:1-13 (2010).

In one type of chimeric antigen receptor (CAR), the variable heavy (VH)and light (VL) chains for a tumor-specific monoclonal antibody are fusedin-frame with the CD3 zeta chain (ζ) from the T cell receptor complex.The VH and VL are generally connected together using a flexibleglycine-serine linker, and then attached to the transmembrane domain bya spacer (CH2CH3) to extend the scFv away from the cell surface so thatit can interact with tumor antigens. Following transduction, T cells nowexpress the CAR on their surface, and upon contact and ligation with atumor antigen, signal through the CD3 zeta chain inducing cytotoxicityand cellular activation.

Investigators have noted that activation of T cells through CD3 zeta issufficient to induce a tumor-specific killing, but is insufficient toinduce T cell proliferation and survival. Early clinical trials using Tcells modified with first generation CARs expressing only the zeta chainshowed that gene-modified T cells exhibited poor survival andproliferation in vivo.

An “antigen recognition moiety” may be any polypeptide or fragmentthereof, such as, for example, an antibody fragment variable domain,either naturally-derived, or synthetic, which binds to an antigen.Examples of antigen recognition moieties include, but are not limitedto, polypeptides derived from antibodies, such as, for example, singlechain variable fragments (scFv), Fab, Fab′, F(ab′)2, and Fv fragments;polypeptides derived from T Cell receptors, such as, for example, TCRvariable domains; and any ligand or receptor fragment that binds to theextracellular cognate protein.

As used herein, the term “cDNA” is intended to refer to DNA preparedusing messenger RNA (mRNA) as template. The advantage of using a cDNA,as opposed to genomic DNA or DNA polymerized from a genomic, non- orpartially processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There are times when thefull or partial genomic sequence is used, such as where the non-codingregions are required for optimal expression or where non-coding regionssuch as introns are to be targeted in an antisense strategy.

As used herein, the term “expression construct” or “transgene” isdefined as any type of genetic construct containing a nucleic acidcoding for gene products in which part or all of the nucleic acidencoding sequence is capable of being transcribed can be inserted intothe vector. The transcript is translated into a protein, but it need notbe. In certain embodiments, expression includes both transcription of agene and translation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encodinggenes of interest. The term “therapeutic construct” may also be used torefer to the expression construct or transgene. The expression constructor transgene may be used, for example, as a therapy to treathyperproliferative diseases or disorders, such as cancer, thus theexpression construct or transgene is a therapeutic construct or aprophylactic construct.

As used herein, the term “expression vector” refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are discussed infra.

By “constitutively active” is meant that the chimeric stimulatingmolecule's T cell activation activity, as demonstrated herein, is activein the absence of an inducer. Constitutively active chimeric stimulatingmolecules in the present application do not comprise a multimeric ligandbinding region, or a functional multimeric ligand binding region, andare not inducible by AP1903, AP20187, or other CID.

As used herein, the term “ex vivo” refers to “outside” the body. Theterms “ex vivo” and “in vitro” can be used interchangeably herein.

As used herein, the term “functionally equivalent,” as it relates toCD40, for example, refers to a CD40 nucleic acid fragment, variant, oranalog, refers to a nucleic acid that codes for a CD40 polypeptide, or aCD40 polypeptide, that stimulates an immune response to destroy tumorsor hyperproliferative disease. “Functionally equivalent” or “afunctional fragment” of a CD40 polypeptide refers, for example, to aCD40 polypeptide that is lacking the extracellular domain, but iscapable of amplifying the T cell-mediated tumor killing response byupregulating dendritic cell expression of antigen presentationmolecules. When the term “functionally equivalent” is applied to othernucleic acids or polypeptides, such as, for example, PSA peptide, PSMApeptide, MyD88, or truncated MyD88, it refers to fragments, variants,and the like that have the same or similar activity as the referencepolypeptides of the methods herein. For example, a functional fragmentof a tumor antigen polypeptide, such as, for example, PSMA may beantigenic, allowing for antibodies to be produced that recognize theparticular tumor antigen. A functional fragment of a ligand bindingregion, for example, Fvls, would include a sufficient portion of theligand binding region polypeptide to bind the appropriate ligand.“Functionally equivalent” refers, for example, to a co-stimulatorypolypeptide that is lacking the extracellular domain, but is capable ofamplifying the T cell-mediated tumor killing response when expressed inT cells.

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occlusion,restenosis, atherosclerosis, or inflammatory bowel disease.

As used herein, the term “gene” is defined as a functional protein,polypeptide, or peptide-encoding unit. As will be understood, thisfunctional term includes genomic sequences, cDNA sequences, and smallerengineered gene segments that express, or are adapted to express,proteins, polypeptides, domains, peptides, fusion proteins, and mutants.

The term “immunogenic composition” or “immunogen” refers to a substancethat is capable of provoking an immune response. Examples of immunogensinclude, e.g., antigens, autoantigens that play a role in induction ofautoimmune diseases, and tumor-associated antigens expressed on cancercells.

The term “immunocompromised” as used herein is defined as a subject thathas reduced or weakened immune system. The immunocompromised conditionmay be due to a defect or dysfunction of the immune system or to otherfactors that heighten susceptibility to infection and/or disease.Although such a categorization allows a conceptual basis for evaluation,immunocompromised individuals often do not fit completely into one groupor the other. More than one defect in the body's defense mechanisms maybe affected. For example, individuals with a specific T-lymphocytedefect caused by HIV may also have neutropenia caused by drugs used forantiviral therapy or be immunocompromised because of a breach of theintegrity of the skin and mucous membranes. An immunocompromised statecan result from indwelling central lines or other types of impairmentdue to intravenous drug abuse; or be caused by secondary malignancy,malnutrition, or having been infected with other infectious agents suchas tuberculosis or sexually transmitted diseases, e.g., syphilis orhepatitis.

As used herein, the term “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells presented herein, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions. In someembodiments, the subject is a mammal. In some embodiments, the subjectis a human.

As used herein, the term “polynucleotide” is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. Nucleic acids are polynucleotides, which can behydrolyzed into the monomeric “nucleotides.” The monomeric nucleotidescan be hydrolyzed into nucleosides. As used herein polynucleotidesinclude, but are not limited to, all nucleic acid sequences which areobtained by any means available in the art, including, withoutlimitation, recombinant means, i.e., the cloning of nucleic acidsequences from a recombinant library or a cell genome, using ordinarycloning technology and PCR™, and the like, and by synthetic means.Furthermore, polynucleotides include mutations of the polynucleotides,include but are not limited to, mutation of the nucleotides, ornucleosides by methods well known in the art. A nucleic acid maycomprise one or more polynucleotides.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide may be interchangeable with the term “proteins”.

As used herein, the term “promoter” is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene.

As used herein, the terms “regulate an immune response,” “modulate animmune response,” or “control an immune response,” refer to the abilityto modify the immune response. For example, the composition is capableof enhancing and/or activating the immune response. Still further, thecomposition is also capable of inhibiting the immune response. The formof regulation is determined by the ligand that is used with thecomposition. For example, a dimeric analog of the chemical results indimerization of the co-stimulating polypeptide leading to activation ofthe T cell, however, a monomeric analog of the chemical does not resultin dimerization of the co-stimulating polypeptide, which would notactivate the T cells.

The term “transfection” and “transduction” are interchangeable and referto the process by which an exogenous DNA sequence is introduced into aeukaryotic host cell. Transfection (or transduction) can be achieved byany one of a number of means including electroporation, microinjection,gene gun delivery, retroviral infection, lipofection, superfection andthe like.

As used herein, the term “syngeneic” refers to cells, tissues or animalsthat have genotypes that are identical or closely related enough toallow tissue transplant, or are immunologically compatible. For example,identical twins or animals of the same inbred strain. Syngeneic andisogeneic can be used interchangeably.

The term “terms “patient” or “subject”” are interchangeable, and, asused herein include, but are not limited to, an organism or animal; amammal, including, e.g., a human, non-human primate (e.g., monkey),mouse, pig, cow, goat, rabbit, rat, guinea pig, hamster, horse, monkey,sheep, or other non-human mammal; a non-mammal, including, e.g., anon-mammalian vertebrate, such as a bird (e.g., a chicken or duck) or afish, and a non-mammalian invertebrate.

As used herein, the term “vaccine” refers to a formulation that containsa composition presented herein which is in a form that is capable ofbeing administered to an animal. Typically, the vaccine comprises aconventional saline or buffered aqueous solution medium in which thecomposition is suspended or dissolved. In this form, the composition canbe used conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a subject, the vaccine is able toprovoke an immune response including, but not limited to, the productionof antibodies, cytokines and/or other cellular responses.

As used herein, the term “under transcriptional control” or “operativelylinked” is defined as the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

By “T cell activation molecule” is meant a polypeptide that, whenincorporated into a T cell expressing a chimeric antigen receptor,enhances activation of the T cell. Examples include, but are not limitedto, ITAM-containing, Signal 1 conferring molecules such as, for example,CD3ζ polypeptide, and Fc receptor gamma, such as, for example, Fcepsilon receptor gamma (FcεR1γ) subunit (Haynes, N. M., et al. J.Immunol. 166:182-7 (2001)). J. Immunology).

As used herein, the terms “treatment”, “treat”, “treated”, or “treating”refer to prophylaxis and/or therapy. When used with respect to a solidtumor, such as a cancerous solid tumor, for example, the term refers toprevention by prophylactic treatment, which increases the subject'sresistance to solid tumors or cancer. In some examples, the subject maybe treated to prevent cancer, where the cancer is familial, or isgenetically associated. When used with respect to an infectious disease,for example, the term refers to a prophylactic treatment which increasesthe resistance of a subject to infection with a pathogen or, in otherwords, decreases the likelihood that the subject will become infectedwith the pathogen or will show signs of illness attributable to theinfection, as well as a treatment after the subject has become infectedin order to fight the infection, for example, reduce or eliminate theinfection or prevent it from becoming worse.

The methods provided herein may be used, for example, to treat adisease, disorder, or condition wherein there is an elevated expressionof a tumor antigen.

As used herein, the term “vaccine” refers to a formulation whichcontains a composition presented herein which is in a form that iscapable of being administered to an animal. Typically, the vaccinecomprises a conventional saline or buffered aqueous solution medium inwhich the composition is suspended or dissolved. In this form, thecomposition can be used conveniently to prevent, ameliorate, orotherwise treat a condition. Upon introduction into a subject, thevaccine is able to provoke an immune response including, but not limitedto, the production of antibodies, cytokines and/or other cellularresponses.

Blood disease: The terms “blood disease”, “blood disease” and/or“diseases of the blood” as used herein, refers to conditions that affectthe production of blood and its components, including but not limitedto, blood cells, hemoglobin, blood proteins, the mechanism ofcoagulation, production of blood, production of blood proteins, the likeand combinations thereof. Non-limiting examples of blood diseasesinclude anemias, leukemias, lymphomas, hematological neoplasms,albuminemias, haemophilias and the like.

Bone marrow disease: The term “bone marrow disease” as used herein,refers to conditions leading to a decrease in the production of bloodcells and blood platelets. In some bone marrow diseases, normal bonemarrow architecture can be displaced by infections (e.g., tuberculosis)or malignancies, which in turn can lead to the decrease in production ofblood cells and blood platelets. Non-limiting examples of bone marrowdiseases include leukemias, bacterial infections (e.g., tuberculosis),radiation sickness or poisoning, apnocytopenia, anemia, multiple myelomaand the like.

T cells and Activated T cells: T cells (also referred to as Tlymphocytes) belong to a group of white blood cells referred to aslymphocytes. Lymphocytes generally are involved in cell-mediatedimmunity. The “T” in “T cells” refers to cells derived from or whosematuration is influenced by the thymus. T cells can be distinguishedfrom other lymphocytes types such as B cells and Natural Killer (NK)cells by the presence of cell surface proteins known as T cellreceptors. The term “activated T cells” as used herein, refers to Tcells that have been stimulated to produce an immune response (e.g.,clonal expansion of activated T cells) by recognition of an antigenicdeterminant presented in the context of a Class II majorhisto-compatibility (MHC) marker. T-cells are activated by the presenceof an antigenic determinant, cytokines and/or lymphokines and cluster ofdifferentiation cell surface proteins (e.g., CD3, CD4, CD8, the like andcombinations thereof). Cells that express a cluster of differentialprotein often are said to be “positive” for expression of that proteinon the surface of T-cells (e.g., cells positive for CD3 or CD 4expression are referred to as CD3⁺ or CD4⁺). CD3 and CD4 proteins arecell surface receptors or co-receptors that may be directly and/orindirectly involved in signal transduction in T cells. Peripheral blood:The term “peripheral blood” as used herein, refers to cellularcomponents of blood (e.g., red blood cells, white blood cells andplatelets), which are obtained or prepared from the circulating pool ofblood and not sequestered within the lymphatic system, spleen, liver orbone marrow.

Umbilical cord blood: Umbilical cord blood is distinct from peripheralblood and blood sequestered within the lymphatic system, spleen, liveror bone marrow. The terms “umbilical cord blood”, “umbilical blood” or“cord blood”, which can be used interchangeably, refers to blood thatremains in the placenta and in the attached umbilical cord after childbirth. Cord blood often contains stem cells including hematopoieticcells.

By “obtained or prepared” as, for example, in the case of cells, ismeant that the cells or cell culture are isolated, purified, orpartially purified from the source, where the source may be, forexample, umbilical cord blood, bone marrow, or peripheral blood. Theterms may also apply to the case where the original source, or a cellculture, has been cultured and the cells have replicated, and where theprogeny cells are now derived from the original source.

By “kill” or “killing” as in a percent of cells killed, is meant thedeath of a cell through apoptosis, as measured using any method knownfor measuring apoptosis. The term may also refer to cell ablation.

Donor T cell: The term “donor T cell” as used here refers to T cellsthat often are administered to a recipient to confer anti-viral and/oranti-tumor immunity following allogeneic stem cell transplantation.Donor T cells often are utilized to inhibit marrow graft rejection andincrease the success of alloengraftment, however the same donor T cellscan cause an alloaggressive response against host antigens, which inturn can result in graft versus host disease (GvHD). Certain activateddonor T cells can cause a higher or lower GvHD response than otheractivated T cells. Donor T cells may also be reactive against recipienttumor cells, causing a beneficial graft vs. tumor effect.

Function-conservative variants are proteins or enzymes in which a givenamino acid residue has been changed without altering overallconformation and function of the protein or enzyme, including, but notlimited to, replacement of an amino acid with one having similarproperties, including polar or non-polar character, size, shape andcharge. Conservative amino acid substitutions for many of the commonlyknown non-genetically encoded amino acids are well known in the art.Conservative substitutions for other non-encoded amino acids can bedetermined based on their physical properties as compared to theproperties of the genetically encoded amino acids.

Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and canbe, for example, at least 70%, for example, at least 80%, for example,at least 90%, and for example, at least 95%, as determined according toan alignment scheme. As referred to herein, “sequence similarity” meansthe extent to which nucleotide or protein sequences are related. Theextent of similarity between two sequences can be based on percentsequence identity and/or conservation. “Sequence identity” herein meansthe extent to which two nucleotide or amino acid sequences areinvariant. “Sequence alignment” means the process of lining up two ormore sequences to achieve maximal levels of identity (and, in the caseof amino acid sequences, conservation) for the purpose of assessing thedegree of similarity. Numerous methods for aligning sequences andassessing similarity/identity are known in the art such as, for example,the Cluster Method, wherein similarity is based on the MEGALIGNalgorithm, as well as BLASTN, BLASTP, and FASTA. When using any of theseprograms, the settings used in certain embodiments are those thatresults in the highest sequence similarity.

Mesenchymal stromal cell: The terms “mesenchymal stromal cell” or “bonemarrow derived mesenchymal stromal cell” as used herein, refer tomultipotent stem cells that can differentiate ex vivo, in vitro and invivo into adipocytes, osteoblasts and chondroblasts, and may be furtherdefined as a fraction of mononuclear bone marrow cells that adhere toplastic culture dishes in standard culture conditions, are negative forhematopoietic lineage markers and are positive for CD73, CD90 and CD105.

Embryonic stem cell: The term “embryonic stem cell” as used herein,refers to pluripotent stem cells derived from the inner cell mass of theblastocyst, an early stage embryo of between 50 to 150 cells. Embryonicstem cells are characterized by their ability to renew themselvesindefinitely and by their ability to differentiate into derivatives ofall three primary germ layers, ectoderm, endoderm and mesoderm.Pluripotent is distinguished from multipotent in that pluripotent cellscan generate all cell types, while multipotent cells (e.g., adult stemcells) can only produce a limited number of cell types.

Inducible pluripotent stem cell: The terms “inducible pluripotent stemcell” or “induced pluripotent stem cell” as used herein refers to adult,or differentiated cells, that are “reprogrammed” or induced by genetic(e.g., expression of genes that in turn activates pluripotency),biological (e.g., treatment viruses or retroviruses) and/or chemical(e.g., small molecules, peptides and the like) manipulation to generatecells that are capable of differentiating into many if not all celltypes, like embryonic stem cells. Inducible pluripotent stem cells aredistinguished from embryonic stem cells in that they achieve anintermediate or terminally differentiated state (e.g., skin cells, bonecells, fibroblasts, and the like) and then are induced todedifferentiate, thereby regaining some or all of the ability togenerate multipotent or pluripotent cells.

CD34⁺ cell: The term “CD34⁺ cell” as used herein refers to a cellexpressing the CD34 protein on its cell surface. “CD34” as used hereinrefers to a cell surface glycoprotein (e.g., sialomucin protein) thatoften acts as a cell-cell adhesion factor and is involved in T cellentrance into lymph nodes, and is a member of the “cluster ofdifferentiation” gene family. CD34 also may mediate the attachment ofstem cells to bone marrow, extracellular matrix or directly to stromalcells. CD34⁺ cells often are found in the umbilical cord and bone marrowas hematopoietic cells, a subset of mesenchymal stem cells, endothelialprogenitor cells, endothelial cells of blood vessels but not lymphatics(except pleural lymphatics), mast cells, a sub-population of dendriticcells (which are factor XIIIa negative) in the interstitium and aroundthe adnexa of dermis of skin, as well as cells in certain soft tissuetumors (e.g., alveolar soft part sarcoma, pre-B acute lymphoblasticleukemia (Pre-B-ALL), acute myelogenous leukemia (AML), AML-M7,dermatofibrosarcoma protuberans, gastrointestinal stromal tumors, giantcell fibroblastoma, granulocytic sarcoma, Kaposi's sarcoma, liposarcoma,malignant fibrous histiocytoma, malignant peripheral nerve sheathtumors, mengingeal hemangiopericytomas, meningiomas, neurofibromas,schwannomas, and papillary thyroid carcinoma).

Tumor infiltrating lymphocytes (TILs) refer to T cells having variousreceptors which infiltrate tumors and kill tumor cells in a targetedmanor. Regulating the activity of the TILs using the methods of thepresent application would allow for more direct control of theelimination of tumor cells.

Gene expression vector: The terms “gene expression vector”, “nucleicacid expression vector”, or “expression vector” as used herein, whichcan be used interchangeably throughout the document, generally refers toa nucleic acid molecule (e.g., a plasmid, phage, autonomouslyreplicating sequence (ARS), artificial chromosome, yeast artificialchromosome (e.g., YAC)) that can be replicated in a host cell and beutilized to introduce a gene or genes into a host cell. The genesintroduced on the expression vector can be endogenous genes (e.g., agene normally found in the host cell or organism) or heterologous genes(e.g., genes not normally found in the genome or on extra-chromosomalnucleic acids of the host cell or organism). The genes introduced into acell by an expression vector can be native genes or genes that have beenmodified or engineered. The gene expression vector also can beengineered to contain 5′ and 3′ untranslated regulatory sequences thatsometimes can function as enhancer sequences, promoter regions and/orterminator sequences that can facilitate or enhance efficienttranscription of the gene or genes carried on the expression vector. Agene expression vector sometimes also is engineered for replicationand/or expression functionality (e.g., transcription and translation) ina particular cell type, cell location, or tissue type. Expressionvectors sometimes include a selectable marker for maintenance of thevector in the host or recipient cell.

Developmentally regulated promoter: The term “developmentally regulatedpromoter” as used herein refers to a promoter that acts as the initialbinding site for RNA polymerase to transcribe a gene which is expressedunder certain conditions that are controlled, initiated by or influencedby a developmental program or pathway. Developmentally regulatedpromoters often have additional control regions at or near the promoterregion for binding activators or repressors of transcription that caninfluence transcription of a gene that is part of a development programor pathway. Developmentally regulated promoters sometimes are involvedin transcribing genes whose gene products influence the developmentaldifferentiation of cells.

Developmentally differentiated cells: The term “developmentallydifferentiated cells”, as used herein refers to cells that haveundergone a process, often involving expression of specificdevelopmentally regulated genes, by which the cell evolves from a lessspecialized form to a more specialized form in order to perform aspecific function. Non-limiting examples of developmentallydifferentiated cells are liver cells, lung cells, skin cells, nervecells, blood cells, and the like. Changes in developmentaldifferentiation generally involve changes in gene expression (e.g.,changes in patterns of gene expression), genetic re-organization (e.g.,remodeling or chromatin to hide or expose genes that will be silenced orexpressed, respectively), and occasionally involve changes in DNAsequences (e.g., immune diversity differentiation). Cellulardifferentiation during development can be understood as the result of agene regulatory network. A regulatory gene and its cis-regulatorymodules are nodes in a gene regulatory network that receive input (e.g.,protein expressed upstream in a development pathway or program) andcreate output elsewhere in the network (e.g., the expressed gene productacts on other genes downstream in the developmental pathway or program).

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occlusion,restenosis, atherosclerosis, or inflammatory bowel disease.

In some embodiments, the nucleic acid is contained within a viralvector. In certain embodiments, the viral vector is an adenoviralvector, or a retroviral or lentiviral vector. It is understood that insome embodiments, the cell is contacted with the viral vector ex vivo,and in some embodiments, the cell is contacted with the viral vector invivo.

In certain embodiments, the cell is also contacted with an antigen.Often, the cell is contacted with the antigen ex vivo. Sometimes, thecell is contacted with the antigen in vivo. In some embodiments, thecell is in a subject and an immune response is generated against theantigen. Sometimes, the immune response is a cytotoxic T-lymphocyte(CTL) immune response. Sometimes, the immune response is generatedagainst a tumor antigen. In certain embodiments, the cell is activatedwithout the addition of an adjuvant.

In some embodiments, the cell is transduced with the nucleic acid exvivo and administered to the subject by intradermal administration. Insome embodiments, the cell is transduced with the nucleic acid ex vivoand administered to the subject by subcutaneous administration.Sometimes, the cell is transduced with the nucleic acid ex vivo.Sometimes, the cell is transduced with the nucleic acid in vivo.

By MyD88, or MyD88 polypeptide, is meant the polypeptide product of themyeloid differentiation primary response gene 88, for example, but notlimited to the human version, cited as ncbi Gene ID 4615. One example ofa MyD88 polypeptide is presented as SEQ ID NO: 282. By “truncated,” ismeant that the protein is not full length and may lack, for example, adomain. For example, a truncated MyD88 is not full length and may, forexample, be missing the TIR domain. One example of a truncated MyD88 isindicated as MyD88L herein, and is also presented as SEQ ID NOS: 5(nucleic acid sequence) and 6 (peptide sequence). SEQ ID NO: 5 includesthe linkers added during subcloning. By a nucleic acid sequence codingfor “truncated MyD88” is meant the nucleic acid sequence coding for thetruncated MyD88 peptide, the term may also refer to the nucleic acidsequence including the portion coding for any amino acids added as anartifact of cloning, including any amino acids coded for by the linkers.It is understood that where a method or construct refers to a truncatedMyD88 polypeptide, the method may also be used, or the constructdesigned to refer to another MyD88 polypeptide, such as a full lengthMyD88 polypeptide. Where a method or construct refers to a full lengthMyD88 polypeptide, the method may also be used, or the constructdesigned to refer to a truncated MyD88 polypeptide.

In the methods herein, the CD40 portion of the peptide may be locatedeither upstream or downstream from the MyD88 or truncated MyD88polypeptide portion.

The cell in some embodiments is contacted with an antigen, sometimes exvivo. In certain embodiments the cell is in a subject and an immuneresponse is generated against the antigen, such as a cytotoxicT-lymphocyte (CTL) immune response. In certain embodiments, an immuneresponse is generated against a tumor antigen (e.g., PSMA). In someembodiments, the nucleic acid is prepared ex vivo and administered tothe subject by intradermal administration or by subcutaneousadministration, for example. Sometimes the cell is transduced ortransfected with the nucleic acid ex vivo or in vivo.

In some embodiments, the nucleic acid comprises a promoter sequenceoperably linked to the polynucleotide sequence. Alternatively, thenucleic acid comprises an ex vivo-transcribed RNA, containing theprotein-coding region of the chimeric protein.

By “reducing tumor size” or “inhibiting tumor growth” of a solid tumoris meant a response to treatment, or stabilization of disease, accordingto standard guidelines, such as, for example, the Response EvaluationCriteria in Solid Tumors (RECIST) criteria. For example, this mayinclude a reduction in the diameter of a solid tumor of about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or the reduction in thenumber of tumors, circulating tumor cells, or tumor markers, of about5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The size oftumors may be analyzed by any method, including, for example, CT scan,MRI, for example, CT-MRI, chest X-ray (for tumors of the lung), ormolecular imaging, for example, PET scan, such as, for example, a PETscan after administering an iodine 123-labelled PSA, for example, PSMAligand, such as, for example, where the inhibitor isTROFEX™/MIP-1072/1095, or molecular imaging, for example, SPECT, or aPET scan using PSA, for example, PSMA antibody, such as, for example,capromad pendetide (Prostascint), a 111-iridium labeled PSMA antibody.

By “reducing, slowing, or inhibiting tumor vascularization” is meant areduction in tumor vascularization of about 5%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100%, or a reduction in the appearance of newvasculature of about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%, when compared to the amount of tumor vascularization beforetreatment. The reduction may refer to one tumor, or may be a sum or anaverage of the vascularization in more than one tumor. Methods ofmeasuring tumor vascularization include, for example, CAT scan, MRI, forexample, CT-MRI, or molecular imaging, for example, SPECT, or a PETscan, such as, for example, a PET scan after administering an iodine123-labelled PSA, for example, PSMA ligand, such as, for example, wherethe inhibitor is TROFEX™/MIP-1072/1095, or a PET scan using PSA, forexample, PSMA antibody, such as, for example, capromad pendetide(Prostascint), a 111-iridium labeled PSMA antibody.

A tumor is classified, or named as part of an organ, such as a prostatecancer tumor when, for example, the tumor is present in the prostategland, or has derived from or metastasized from a tumor in the prostategland, or produces PSA. A tumor has metastasized from a tumor in theprostate gland, when, for example, it is determined that the tumor haschromosomal breakpoints that are the same as, or similar to, a tumor inthe prostate gland of the subject.

Prostate Cancer

In the United States, prostate cancer is the most common solid tumormalignancy in men. It was expected to account for an estimated 186,320new cases of prostate cancer in 2008 and 28,660 deaths. Jemal A, et al.,Cancer statistics, 2008. CA Cancer J Clin. 58: 71-96, 2008.Approximately 70% of patients who experience PSA-progression afterprimary therapy will have metastases at some time during the course oftheir disease. Gittes R F, N Engl J Med. 324: 236-45, 1991. Androgendeprivation therapy (ADT) is the standard therapy for metastaticprostate cancer and achieves temporary tumor control or regression in80-85% of patients. Crawford E D, et al., N Engl J Med. 321: 419-24,1989; Schellhammer P F, et al., J Urol. 157: 1731-5, 1997; Scher H I andKelly W K, J Clin Oncol. 11: 1566-72, 1993; Small E J and Srinivas S,Cancer. 76: 1428-34, 1995.

Duration of response to hormone therapy, as well as survival after theinitiation of hormone therapy, has been shown to be dependent on anumber of factors, including the Gleason Sum of the original tumor, theability to achieve an undetectable nadir PSA after initiation of ADT,and the PSA doubling time prior to initiation of ADT. Despite hormonaltherapy, virtually all patients with metastatic prostate cancerultimately develop progressive disease. Kelly W K and Slovin S F, CurrOncol Rep. 2: 394-401, 2000; Scher H I, et al., J Natl Cancer Inst. 88:1623-34, 1996; Small E J and Vogelzang N.J., J Clin Oncol. 15: 382-8,1997. The Gleason Sum of the original tumor, or the Gleason score, isused to grade levels of prostate cancer in men, based on the microscopicevaluation of the tumor. A higher Gleason score denotes a cancer thathas a worse prognosis as it is more aggressive, and is more likely tospread. An example of the grading system is discussed in Gleason D F.,The Veteran's Administration Cooperative Urologic Research Group:histologic grading and clinical staging of prostatic carcinoma. InTannenbaum M (ed.) Urologic Pathology: The Prostate. Lea and Febiger,Philadelphia, 1977; 171-198.

Most patients with prostate cancer who have been started on ADT aretreated for a rising PSA after failure of primary therapy (e.g. radicalprostatectomy, brachytherapy, external beam radiation therapy,cryo-ablation, etc.). In the absence of clinical metastases, thesepatients experience a relatively long disease-free interval in the rangeof 7-11 years; however, the majority of these patients eventuallydevelop hormone-resistant disease as evidenced by the return of a risingPSA level in the face of castrate levels of serum testosterone. Thesepatients, too, have a poor prognosis, with the majority developingclinical metastases within 9 months and a median survival of 24 months.Bianco F J, et al., Cancer Symposium: Abstract 278, 2005. The term“prostate cancer” includes different forms or stages, including, forexample, metastatic, metastatic castration resistant, metastaticcastration sensitive, regionally advanced, and localized prostatecancer.

Engineering Expression Constructs

Expression constructs that express the present chimeric stimulatingmolecules comprise the chimeric stimulating molecule coding region and apromoter sequence, all operatively linked. Expression constructs thatexpress the present MyD88/CD40-encoding chimeric antigen receptorscomprise the MyD88/CD40 chimeric antigen receptor coding region and apromoter sequence, all operatively linked. In general, the term“operably linked” is meant to indicate that the promoter sequence isfunctionally linked to a second sequence, wherein the promoter sequenceinitiates and mediates transcription of the DNA corresponding to thesecond sequence.

In certain examples, the polynucleotide coding for the chimericstimulating molecule or the MyD88/CD40 chimeric antigen receptor codingregion is included in the same vector, such as, for example, a viral orplasmid vector, as a polynucleotide coding for the second polypeptide.This second polypeptide may be, for example, a caspase polypeptide, asdiscussed herein, or a marker polypeptide. Where the vector expresses achimeric stimulating molecule, the second polypeptide may also be, forexample, a non-MyD88/CD40-containing chimeric antigen receptor. In theseexamples, the construct may be designed with one promoter operablylinked to a nucleic acid comprising a polynucleotide coding for the twopolypeptides, linked by a cleavable 2A polypeptide. In this example, thefirst and second polypeptides are separated during translation,resulting in either a chimeric stimulating molecule or a MyD88/CD40chimeric antigen receptor polypeptide, and the second polypeptide. Inother examples, the two polypeptides may be expressed separately fromthe same vector, where each nucleic acid comprising a polynucleotidecoding for one of the polypeptides is operably linked to a separatepromoter. In yet other examples, one promoter may be operably linked tothe two polynucleotides, directing the production of two separate RNAtranscripts, and thus two polypeptides; in one example, the promoter maybe bi-directional, and the coding regions may be in opposite directions5′-3′. Therefore, the expression constructs discussed herein maycomprise at least one, or at least two promoters.

In yet other examples, two polypeptides, such as, for example, thechimeric stimulating molecule or a MyD88/CD40 chimeric antigen receptorpolypeptide, and the second polypeptide, may be expressed in a cellusing two separate vectors. The cells may be co-transfected orco-transformed with the vectors, or the vectors may be introduced to thecells at different times.

The polypeptides may vary in their order, from the amino terminus to thecarboxy terminus. For example, in the chimeric stimulating molecule, theorder of the MyD88 polypeptide, CD40 polypeptide, and any additionalpolypeptide, may vary. In the chimeric antigen receptor, the order ofthe MyD88 polypeptide, CD40 polypeptide, and any additional polypeptide,such as, for example, the CD3ζ polypeptide may vary. The order of thevarious domains may be assayed using methods such as, for example, thosediscussed herein, to obtain the optimal expression and activity.

In certain embodiments, a nucleic acid molecule is provided thatcomprises a promoter operably linked to a first polynucleotide encodinga chimeric stimulating molecule, wherein the chimeric stimulatingmolecule comprises (i) a MyD88 polypeptide or a truncated MyD88polypeptide lacking the TIR domain; and (ii) a CD40 cytoplasmicpolypeptide region lacking the CD40 extracellular domain, and whereinthe chimeric stimulating molecule does not include a membrane targetingregion; and

-   -   b) a second polynucleotide encoding a T cell receptor, a T cell        receptor-based chimeric antigen receptor, or a chimeric antigen        receptor; and    -   c) a third polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide. It is understood that the order of the        polynucleotides may vary, and may be tested to determine the        suitability of the construct for any particular method, thus,        the nucleic acid may include the polynucleotides in the varying        orders, which also take into account a variation in the order of        the MyD88 polypeptide or truncated MyD88 polypeptide-encoding        sequence and the CD40 cytoplasmic polypeptide region-encoding        sequence in the first polynucleotide. Thus the first        polynucleotide may encode a polypeptide having and order of        MyD88/CD40, truncated MyD88/CD40, CD40/MyD88, or CD40/truncated        MyD88. And, the nucleic acid may include the first through third        polynucleotides in any of the following orders, where 1, 2, 3,        indicate a first, second, or third order of the polynucleotide        in the nucleic acid from 5′ to 3′. It is understood that other        polynucleotides, such as those that code for a 2A polypeptide,        for example, may be present between the three listed        polynucleotides; this Table is meant to designate the order of        the first through third polynucleotides:

TABLE A Second First polynucleotide polynucleotide encoding a encoding aT cell Chimeric stimulating receptor, a T cell molecule comprisingreceptor-based Third MyD88 or truncated chimeric antigen polynucleotideMyD88 and CD40 receptor, or a encoding a cytoplasmic polypeptidechimeric antigen chimeric caspse-9 region. receptor. polypeptide. 1 2 31 3 2 2 1 3 3 1 2 2 3 1 3 2 1

Similarly, the nucleic acids may include only two of thepolynucleotides, coding for two of the polypeptides provided in thetable above. In some examples, a cell is transfected or transduced witha nucleic acid comprising the three polynucleotides included in Table Aabove. In other examples, a cell is transfected or transduced with anucleic acid that encodes two of the polynucleotides, coding for two ofthe polypeptides, as provided, for example, in Table B.

TABLE B Second First polynucleotide polynucleotide encoding a encoding aT cell Chimeric stimulating receptor, a T cell molecule comprisingreceptor-based Third MyD88 or truncated chimeric antigen polynucleotideMyD88 and CD40 receptor, or a encoding a cytoplasmic polypeptidechimeric antigen chimeric caspse-9 region. receptor. polypeptide. 1 2 12 2 1 1 2 2 1 2 1

In some embodiments, the cell is transfected or transduced with thenucleic acid that encodes two of the polynucleotides, and the cell alsocomprises a nucleic acid comprising a polynucleotide coding for thethird polypeptide. For example, a cell may comprise a nucleic acidcomprising the first and second polynucleotides, and the cell may alsocomprise a nucleic acid comprising a polynucleotide coding for achimeric Caspase-9 polypeptide. Also, a cell may comprise a nucleic acidcomprising the first and third polynucleotides, and the cell may alsocomprise a nucleic acid comprising a polynucleotide coding for a T cellreceptor, a T cell receptor-based chimeric antigen receptor, or achimeric antigen receptor.

T cell receptors are molecules composed of two different polypeptidesthat are on the surface of T cells. They recognize antigens bound tomajor histocompatibility complex molecules; upon recognition with theantigen, the T cell is activated. By “recognize” is meant, for example,that the T cell receptor, or fragment or fragments thereof, such as TCRαpolypeptide and TCRβ together, is capable of contacting the antigen andidentifying it as a target. TCRs may comprise a and β polypeptides, orchains. The α and β polypeptides include two extracellular domains, thevariable and the constant domains. The variable domain of the α and βpolypeptides has three complementarity determining regions (CDRs); CDR3is considered to be the main CDR responsible for recognizing theepitope. The α polypeptide includes the V and J regions, generated by VJrecombination, and the β polypeptide includes the V, D, and J regions,generated by VDJ recombination. The intersection of the VJ regions andVDJ regions corresponds to the CDR3 region. TCRs are often named usingthe International Immunogenetics (IMGT) TCR nomenclature (IMGT Database,www.IMGT.org; Giudicelli, V., et al., IMGT/LIGM-DB, the IMGT®comprehensive database of immunoglobulin and T cell receptor nucleotidesequences, Nucl. Acids Res., 34, D781-D784 (2006). PMID: 16381979; Tcell Receptor Factsbook, LeFranc and LeFranc, Academic Press ISBN0-12-441352-8). T cell receptor-based chimeric antigen receptors, orTCR-like chimeric antigen receptors are chimeric antigen receptors withTCR-like specificity, as discussed in, for example, Zhang, G., er al.,Nature Scientific Reports 4, Article 3571 (2014) and Zhang, G., et al.,Immunol. Cell. Biol 91(10): 615-24 (2013), which are hereby incorporatedby reference herein in their entirety.

The steps of the methods provided may be performed using any suitablemethod; these methods include, without limitation, methods oftransducing, transforming, or otherwise providing nucleic acid to thecell, presented herein. In some embodiments, the truncated MyD88 peptideis encoded by the nucleotide sequence of SEQ ID NO: 5 (with or withoutDNA linkers or has the amino acid sequence of SEQ ID NO: 6). In someembodiments, the CD40 cytoplasmic polypeptide region is encoded by apolynucleotide sequence in SEQ ID NO: 1.

Selectable Markers

In certain embodiments, the expression constructs contain nucleic acidconstructs whose expression is identified in vitro or in vivo byincluding a marker in the expression construct. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression construct. Usually the inclusion of adrug selection marker aids in cloning and in the selection oftransformants. For example, genes that confer resistance to neomycin,puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are usefulselectable markers. Alternatively, enzymes such as Herpes Simplex Virusthymidine kinase (tk) are employed. Immunologic surface markerscontaining the extracellular, non-signaling domains or various proteins(e.g. CD34, CD19, LNGFR) also can be employed, permitting astraightforward method for magnetic or fluorescence antibody-mediatedsorting. The selectable marker employed is not believed to be important,so long as it is capable of being expressed simultaneously with thenucleic acid encoding a gene product. Further examples of selectablemarkers include, for example, reporters such as GFP, EGFP, β-gal orchloramphenicol acetyltransferase (CAT). In certain embodiments, themarker protein, such as, for example, CD19 is used for selection of thecells for transfusion, such as, for example, in immunomagneticselection. As discussed herein, a CD19 marker is distinguished from ananti-CD19 antibody, or, for example, an scFv, TCR, or other antigenrecognition moiety that binds to CD19.

In certain embodiments, the marker polypeptide is linked to theinducible chimeric stimulating molecule. For example, the markerpolypeptide may be linked to the inducible chimeric stimulating moleculevia a polypeptide sequence, such as, for example, a cleavable 2A-likesequence. The marker polypeptide may be, for example, CD19, ΔCD19, ormay be, for example, a heterologous protein, selected to not affect theactivity of the inducible chimeric stimulating molecule.

2A-like sequences, or “peptide bond-skipping” 2A sequences, are derivedfrom, for example, many different viruses, including, for example, fromThosea asigna. These sequences are sometimes also known as “peptideskipping sequences.” When this type of sequence is placed within acistron, between two peptides that are intended to be separated, theribosome appears to skip a peptide bond, in the case of Thosea asignasequence; the bond between the Gly and Pro amino acids at the carboxyterminal “P-G-P” is omitted. This leaves two to three polypeptides, inthis case the co-stimulating polypeptide cytoplasmic region and themarker polypeptide. When this sequence is used, the peptide that isencoded 5′ of the 2A sequence may end up with additional amino acids atthe carboxy terminus, including the Gly residue and any upstreamresidues in the 2A sequence. The peptide that is encoded 3′ of the 2Asequence may end up with additional amino acids at the amino terminus,including the Pro residue and any downstream residues following the 2Asequence.

In some embodiments, a polypeptide may be included in the expressionvector to aid in sorting cells. For example, the CD34 minimal epitopemay be incorporated into the vector. In some embodiments, the expressionvectors used to express the chimeric antigen receptors or chimericstimulating molecules provided herein further comprise a polynucleotidethat encodes the 16 amino acid CD34 minimal epitope. In someembodiments, such as certain embodiments provided in the examplesherein, the CD34 minimal epitope is incorporated at the amino terminalposition of the CD8 stalk.

Co-Stimulating Polypeptides

Co-stimulating polypeptide molecules are capable of amplifying thecell-mediated immune response through activation of signaling pathwaysinvolved in cell survival and proliferation. Co-stimulating proteinsthat are contemplated include, for example, but are not limited, to themembers of tumor necrosis factor receptor (TNFR) family (i.e., CD40,RANK/TRANCE-R, OX40, 4-1BB) and CD28 family members (CD28, ICOS).Co-stimulating proteins may include, for example, CD28, 4-1BB, OX40, andthe CD3ζ chain, or, for example, the cytoplasmic regions thereof. Morethan one co-stimulating polypeptide or co-stimulating polypeptidecytoplasmic region may be used in the inducible chimeric stimulatingmolecules discussed herein.

Co-stimulating polypeptides include any molecule or polypeptide thatactivates the NF-κB pathway, Akt pathway, and/or p38 pathway. Thecellular activation system is based upon utilizing a recombinantsignaling molecule fused to one or more ligand-binding domains (i.e., asmall molecule binding domain) in which the co-stimulating polypeptideis activated and/or regulated with a ligand resulting in oligomerization(i.e., a lipid-permeable, organic, dimerizing drug). Other systems thatmay be used for crosslinking, or for oligomerization, of co-stimulatingpolypeptides include antibodies, natural ligands, and/or artificialcross-reacting or synthetic ligands. Yet further, another dimerizationsystems contemplated include the coumermycin/DNA gyrase B system.

Co-stimulating polypeptides that can be used include those that activateNF-κB and other variable signaling cascades for example the p38 pathwayand/or Akt pathway. Such co-stimulating polypeptides include, but arenot limited to CD28 family members (e.g. CD28, ICOS), TNF receptors(i.e., CD40, RANK/TRANCE-R, OX40, 4-1BB).

In specific embodiments, the co-stimulating polypeptide molecule isCD40, truncated MyD88, or a chimeric truncated MyD88/CD40 polypeptide.

The CD40 molecule comprises a nucleic acid molecule which: (1)hybridizes under stringent conditions to a nucleic acid having thesequence of a known CD40 gene and (2) codes for a CD40 polypeptide. TheCD40 polypeptide may, in certain examples, lack the extracellulardomain. Exemplary polynucleotide sequences that encode CD40 polypeptidesinclude, but are not limited to SEQ. ID. NO: 1 and CD40 isoforms fromother species. It is contemplated that other normal or mutant variantsof CD40 can be used in the present methods and compositions. Thus, aCD40 region can have an amino acid sequence that differs from the nativesequence by one or more amino acid substitutions, deletions and/orinsertions. For example, one or more TNF receptor associated factor(TRAF) binding regions may be eliminated or effectively eliminated(e.g., a CD40 amino acid sequence is deleted or altered such that a TRAFprotein does not bind or binds with lower affinity than it binds to thenative CD40 sequence). In particular embodiments, a TRAF 3 bindingregion is deleted or altered such that it is eliminated or effectivelyeliminated (e.g., amino acids 250-254 may be altered or deleted; Haueret al., PNAS 102(8): 2874-2879 (2005)).

In certain embodiments, the present methods involve the manipulation ofgenetic material to produce expression constructs. Such methods involvethe generation of expression constructs containing, for example, aheterologous nucleic acid sequence encoding the chimeric stimulatingmolecules or chimeric antigen receptors discussed herein, and a meansfor their expression. The vector can be replicated in an appropriatehelper cell, viral particles may be produced therefrom, and cellsinfected with the recombinant virus particles.

In the context of gene therapy, the gene may be a heterologouspolynucleotide sequence derived from a source other than the viralgenome, which provides the backbone of the vector. The gene is derivedfrom a prokaryotic or eukaryotic source such as a bacterium, a virus,yeast, a parasite, a plant, or even an animal. The heterologous DNA alsois derived from more than one source, i.e., a multigene construct or afusion protein. The heterologous DNA also may include a regulatorysequence, which is derived from one source and the gene from a differentsource.

Co-stimulating polypeptides may comprise, but are not limited to, theamino acid sequences provided herein, and may include functionalconservative mutations, including deletions or truncations, and maycomprise amino acid sequences that are 70%, 75%, 80%, 85%, 90%, 95% or100% identical to the amino acid sequences provided herein.

Ligand-Binding Regions

Ligand binding regions may be included in the chimeric polypeptidesdiscussed herein, for example, as part of the inducible caspasepolypeptides. The ligand-binding (“dimerization”) domain of theexpression construct can be any convenient domain that will allow forinduction using a natural or unnatural ligand, for example, an unnaturalsynthetic ligand. The multimerizing region, multimeric ligand bindingregion, multimerizing region, or ligand-binding domain can be internalor external to the cellular membrane, depending upon the nature of theconstruct and the choice of ligand. A wide variety of ligand-bindingproteins, including receptors, are known, including ligand-bindingproteins associated with the cytoplasmic regions indicated above. Asused herein the term “ligand-binding domain can be interchangeable withthe term “receptor”. Of particular interest are ligand-binding proteinsfor which ligands (for example, small organic ligands) are known or maybe readily produced. These ligand-binding domains or receptors includethe FKBPs and cyclophilin receptors, the steroid receptors, thetetracycline receptor, the other receptors indicated above, and thelike, as well as “unnatural” receptors, which can be obtained fromantibodies, particularly the heavy or light chain subunit, mutatedsequences thereof, random amino acid sequences obtained by stochasticprocedures, combinatorial syntheses, and the like. In certainembodiments, the ligand-binding region is selected from the groupconsisting of FKBP ligand-binding region, cyclophilin receptorligand-binding region, steroid receptor ligand-binding region,cyclophilin receptors ligand-binding region, and tetracycline receptorligand-binding region. Often, the ligand-binding region comprises anF_(v)F_(vls) sequence. Sometimes, the F_(v′)f_(vls) sequence furthercomprises an additional Fv′ sequence. Examples include, for example,those discussed in Kopytek, S. J., et al., Chemistry & Biology 7:313-321(2000) and in Gestwicki, J. E., et al., Combinatorial Chem. & HighThroughput Screening 10:667-675 (2007); Clackson T (2006) Chem Biol DrugDes 67:440-2; Clackson, T., in Chemical Biology: From Small Molecules toSystems Biology and Drug Design (Schreiber, s., et al., eds., Wiley,2007)).

For the most part, the ligand-binding domains or receptor domains willbe at least about 50 amino acids, and fewer than about 350 amino acids,usually fewer than 200 amino acids, either as the natural domain ortruncated active portion thereof. The binding domain may, for example,be small (<25 kDa, to allow efficient transfection in viral vectors),monomeric, nonimmunogenic, have synthetically accessible, cellpermeable, nontoxic ligands that can be configured for dimerization.

The receptor domain can be intracellular or extracellular depending uponthe design of the expression construct and the availability of anappropriate ligand. For hydrophobic ligands, the binding domain can beon either side of the membrane, but for hydrophilic ligands,particularly protein ligands, the binding domain will usually beexternal to the cell membrane, unless there is a transport system forinternalizing the ligand in a form in which it is available for binding.For an intracellular receptor, the construct can encode a signal peptideand transmembrane domain 5′ or 3′ of the receptor domain sequence or mayhave a lipid attachment signal sequence 5′ of the receptor domainsequence. Where the receptor domain is between the signal peptide andthe transmembrane domain, the receptor domain will be extracellular.

The portion of the expression construct encoding the receptor can besubjected to mutagenesis for a variety of reasons. The mutagenizedprotein can provide for higher binding affinity, allow fordiscrimination by the ligand of the naturally occurring receptor and themutagenized receptor, provide opportunities to design a receptor-ligandpair, or the like. The change in the receptor can involve changes inamino acids known to be at the binding site, random mutagenesis usingcombinatorial techniques, where the codons for the amino acidsassociated with the binding site or other amino acids associated withconformational changes can be subject to mutagenesis by changing thecodon(s) for the particular amino acid, either with known changes orrandomly, expressing the resulting proteins in an appropriateprokaryotic host and then screening the resulting proteins for binding.

Antibodies and antibody subunits, e.g., heavy or light chain,particularly fragments, more particularly all or part of the variableregion, or fusions of heavy and light chain to create high-affinitybinding, can be used as the binding domain. Antibodies that arecontemplated include ones that are an ectopically expressed humanproduct, such as an extracellular domain that would not trigger animmune response and generally not expressed in the periphery (i.e.,outside the CNS/brain area). Such examples, include, but are not limitedto low affinity nerve growth factor receptor (LNGFR), and embryonicsurface proteins (i.e., carcinoembryonic antigen). Yet further,antibodies can be prepared against haptenic molecules, which arephysiologically acceptable, and the individual antibody subunitsscreened for binding affinity. The cDNA encoding the subunits can beisolated and modified by deletion of the constant region, portions ofthe variable region, mutagenesis of the variable region, or the like, toobtain a binding protein domain that has the appropriate affinity forthe ligand. In this way, almost any physiologically acceptable hapteniccompound can be employed as the ligand or to provide an epitope for theligand. Instead of antibody units, natural receptors can be employed,where the binding domain is known and there is a useful ligand forbinding.

Oligomerization

The transduced signal will normally result from ligand-mediatedoligomerization of the chimeric protein molecules, i.e., as a result ofoligomerization following ligand-binding, although other binding events,for example allosteric activation, can be employed to initiate a signal.The construct of the chimeric protein will vary as to the order of thevarious domains and the number of repeats of an individual domain.

For multimerizing the Caspase-9 polypeptide, the ligand for theligand-binding domains/receptor domains of the chimeric inducibleCaspase-9 polypeptides will usually be multimeric in the sense that itwill have at least two binding sites, with each of the binding sitescapable of binding to the ligand receptor domain. By “multimeric ligandbinding region” is meant a ligand binding region that binds to amultimeric ligand. The term “multimeric ligands” include dimericligands. A dimeric ligand will have two binding sites capable of bindingto the ligand receptor domain. Desirably, the subject ligands will be adimer or higher order oligomer, usually not greater than abouttetrameric, of small synthetic organic molecules, the individualmolecules typically being at least about 150 Da and less than about 5kDa, usually less than about 3 kDa. A variety of pairs of syntheticligands and receptors can be employed. For example, in embodimentsinvolving natural receptors, dimeric FK506 can be used with an FKBP12receptor, dimerized cyclosporin A can be used with the cyclophilinreceptor, dimerized estrogen with an estrogen receptor, dimerizedglucocorticoids with a glucocorticoid receptor, dimerized tetracyclinewith the tetracycline receptor, dimerized vitamin D with the vitamin Dreceptor, and the like. Alternatively higher orders of the ligands,e.g., trimeric can be used. For embodiments involving unnaturalreceptors, e.g., antibody subunits, modified antibody subunits, singlechain antibodies comprised of heavy and light chain variable regions intandem, separated by a flexible linker domain, or modified receptors,and mutated sequences thereof, and the like, any of a large variety ofcompounds can be used. A significant characteristic of these ligandunits is that each binding site is able to bind the receptor with highaffinity and they are able to be dimerized chemically. Also, methods areavailable to balance the hydrophobicity/hydrophilicity of the ligands sothat they are able to dissolve in serum at functional levels, yetdiffuse across plasma membranes for most applications.

In certain embodiments, the present methods utilize the technique ofchemically induced dimerization (CID) to produce a conditionallycontrolled protein or polypeptide. In addition to this technique beinginducible, it also is reversible, due to the degradation of the labiledimerizing agent or administration of a monomeric competitive inhibitor.Examples of CIDs include, but are not limited to, AP1903 and AP20187.

The CID system uses synthetic bivalent ligands to rapidly crosslinksignaling molecules that are fused to ligand-binding domains. Thissystem has been used to trigger the oligomerization and activation ofcell surface (Spencer, D. M., et al., Science, 1993. 262: p. 1019-1024;Spencer D. M. et al., Curr Biol 1996, 6:839-847; Blau, C. A. et al.,Proc Natl Acad. Sci. USA 1997, 94:3076-3081), or cytosolic proteins(Luo, Z. et al., Nature 1996, 383:181-185; MacCorkle, R. A. et al., ProcNatl Acad Sci USA 1998, 95:3655-3660), the recruitment of transcriptionfactors to DNA elements to modulate transcription (Ho, S. N. et al.,Nature 1996, 382:822-826; Rivera, V. M. et al., Nat. Med. 1996,2:1028-1032) or the recruitment of signaling molecules to the plasmamembrane to stimulate signaling (Spencer D. M. et al., Proc. Natl. Acad.Sci. USA 1995, 92:9805-9809; Holsinger, L. J. et al., Proc. Natl. Acad.Sci. USA 1995, 95:9810-9814).

The CID system is based upon the notion that surface receptoraggregation effectively activates downstream signaling cascades. In thesimplest embodiment, the CID system uses a dimeric analog of the lipidpermeable immunosuppressant drug, FK506, which loses its normalbioactivity while gaining the ability to crosslink molecules geneticallyfused to the FK506-binding protein, FKBP12. By fusing one or more FKBPsand a myristoylation sequence to the cytoplasmic signaling domain of atarget receptor, one can stimulate signaling in a dimerizerdrug-dependent, but ligand and ectodomain-independent manner. Thisprovides the system with temporal control, reversibility using monomericdrug analogs, and enhanced specificity. The high affinity ofthird-generation AP20187/AP1903 CIDs for their binding domain, FKBP12permits specific activation of the recombinant receptor in vivo withoutthe induction of non-specific side effects through endogenous FKBP12.FKBP12 variants having amino acid substitutions and deletions, such asFKBP12_(v)36, that bind to a dimerizer drug, may also be used. Inaddition, the synthetic ligands are resistant to protease degradation,making them more efficient at activating receptors in vivo than mostdelivered protein agents.

The ligands used are capable of binding to two or more of theligand-binding domains. The chimeric proteins may be able to bind tomore than one ligand when they contain more than one ligand-bindingdomain. The ligand is typically a non-protein or a chemical. Exemplaryligands include, but are not limited to dimeric FK506 (e.g., FK1012).

Other ligand binding regions may be, for example, dimeric regions, ormodified ligand binding regions with a wobble substitution, such as, forexample, FKBP12(V36): The human 12 kDa FK506-binding protein with an F36to V substitution, the complete mature coding sequence (amino acids1-107), provides a binding site for synthetic dimerizer drug AP1903(Jemal, A. et al., CA Cancer J. Clinic. 58, 71-96 (2008); Scher, H. I.and Kelly, W. K., Journal of Clinical Oncology 11, 1566-72 (1993)). Twotandem copies of the protein may also be used in the construct so thathigher-order oligomers are induced upon cross-linking by AP1903.

F36V′-FKBP: F36V′-FKBP is a codon-wobbled version of F36V-FKBP. Itencodes the identical polypeptide sequence as F36V-FKPB but has only 62%homology at the nucleotide level. F36V′-FKBP was designed to reducerecombination in retroviral vectors (Schellhammer, P. F. et al., J.Urol. 157, 1731-5 (1997)). F36V′-FKBP was constructed by a PCR assemblyprocedure. The transgene contains one copy of F36V′-FKBP linked directlyto one copy of F36V-FKBP.

In some embodiments, the ligand is a small molecule. The appropriateligand for the selected ligand-binding region may be selected. Often,the ligand is dimeric, sometimes, the ligand is a dimeric FK506 or adimeric FK506 analog. In certain embodiments, the ligand is AP1903 (CASIndex Name: 2-Piperidinecarboxylic acid, 1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-, 1,2-ethanediylbis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]]ester, [2S-[1(R*),2R[S[S*[1(R*),2R*]]]]]-(9Cl) CAS Registry Number:195514-63-7; Molecular Formula: C78H98N4O20 Molecular Weight: 1411.65).In certain embodiments, the ligand is AP20187. In certain embodiments,the ligand is an AP20187 analog, such as, for example, AP1510. In someembodiments, certain analogs will be appropriate for the FKBP12, andcertain analogs appropriate for the mutant (V36) version of FKBP12. Incertain embodiments, one ligand binding region is included in thechimeric protein. In other embodiments, two or more ligand bindingregions are included. Where, for example, the ligand binding region isFKBP12, where two of these regions are included, one may, for example,be the wobbled version.

In such methods, the multimeric molecule can be an antibody that bindsto an epitope in the CD40 extracellular domain (e.g., humanizedanti-CD40 antibody; Tai et al., Cancer Research 64, 2846-2852 (2004)),can be a CD40 ligand (e.g., U.S. Pat. No. 6,497,876 (Maraskovsky etal.)) or may be another co-stimulating molecule (e.g., B7/CD28). It isunderstood that conservative variations in sequence, that do not affectthe function, as assayed herein, are within the scope of the presentclaims.

Other dimerization systems contemplated include the coumermycin/DNAgyrase B system. Coumermycin-induced dimerization activates a modifiedRaf protein and stimulates the MAP kinase cascade. See Farrar et al.,1996.

AP1903 API is manufactured by Alphora Research Inc. and AP1903 DrugProduct for Injection is made by AAI Pharma Services Corp. It isformulated as a 5 mg/mL solution of AP1903 in a 25% solution of thenon-ionic solubilizer Solutol H S 15 (250 mg/mL, BASF). At roomtemperature, this formulation is a clear solution. Upon refrigeration,this formulation undergoes a reversible phase transition on extendedstorage, resulting in a milky solution. This phase transition isreversed upon re-warming to room temperature. The fill is 8 mL in a 10mL glass vial (˜40 mg AP1903 for Injection total per vial).

For use, the AP1903 will be warmed to room temperature and diluted priorto administration. For subjects over 50 kg, the AP1903 is administeredvia i.v. infusion at a dose of 40 mg diluted in 100 mL physiologicalsaline over 2 hours at a rate of 50 mL per hour using a DEHP-free salinebag and solution set. Subjects less than 50 kg receive 0.4 mg/kg AP1903.

All study medication is maintained at a temperature between 2 degrees C.and 8 degrees C., protected from excessive light and heat, and stored ina locked area with restricted access.

Upon determining a need to administer AP1903 and activate Caspase-9 inorder to induce apoptosis of the engineered CAR-expressing T cells,patients may be, for example, administered a single fixed dose of AP1903for Injection (0.4 mg/kg) via IV infusion over 2 hours, using anon-DEHP, non-ethylene oxide sterilized infusion set. The dose of AP1903is calculated individually for all patients, and is not be recalculatedunless body weight fluctuates by 0%. The calculated dose is diluted in100 mL in 0.9% normal saline before infusion.

In a previous Phase I study of AP1903, 24 healthy volunteers weretreated with single doses of AP1903 for Injection at dose levels of0.01, 0.05, 0.1, 0.5 and 1.0 mg/kg infused IV over 2 hours. AP1903plasma levels were directly proportional to dose, with mean Cmax valuesranging from approximately 10-1275 ng/mL over the 0.01-1.0 mg/kg doserange. Following the initial infusion period, blood concentrationsdemonstrated a rapid distribution phase, with plasma levels reduced toapproximately 18, 7, and 1% of maximal concentration at 0.5, 2 and 10hours post-dose, respectively. AP1903 for Injection was shown to be safeand well tolerated at all dose levels and demonstrated a favorablepharmacokinetic profile. Iuliucci J D, et al., J Clin Pharmacol. 41:870-9, 2001.

The fixed dose of AP1903 for injection used, for example, may be 0.4mg/kg intravenously infused over 2 hours. The amount of AP1903 needed invitro for effective signaling of cells is about 10-100 nM (MW: 1412 Da).This equates to 14-140 μg/L or ˜0.014-0.14 mg/kg (1.4-140 μg/kg). Thedosage may vary according to the application, and may, in certainexamples, be more in the range of 0.1-10 nM, or in the range of 50-150nM, 10-200 nM, 75-125 nM, 100-500 nM, 100-600 nM, 100-700 nM, 100-800nM, or 100-900 nM. Doses up to 1 mg/kg were well-tolerated in the PhaseI study of AP1903 provided above.

Membrane-Targeting

A membrane-targeting sequence provides for transport of the chimericprotein to the cell surface membrane, where the same or other sequencescan encode binding of the chimeric protein to the cell surface membrane.Molecules in association with cell membranes contain certain regionsthat facilitate the membrane association, and such regions can beincorporated into a chimeric protein molecule to generatemembrane-targeted molecules. For example, some proteins containsequences at the N-terminus or C-terminus that are acylated, and theseacyl moieties facilitate membrane association. Such sequences arerecognized by acyltransferases and often conform to a particularsequence motif. Certain acylation motifs are capable of being modifiedwith a single acyl moiety (often followed by several positively chargedresidues (e.g. human c-Src: M-G-S-N-K-S-K-P-K-D-A-S-Q-R-R-R) to improveassociation with anionic lipid head groups) and others are capable ofbeing modified with multiple acyl moieties. For example the N-terminalsequence of the protein tyrosine kinase Src can comprise a singlemyristoyl moiety. Dual acylation regions are located within theN-terminal regions of certain protein kinases, such as a subset of Srcfamily members (e.g., Yes, Fyn, Lck) and G-protein alpha subunits. Suchdual acylation regions often are located within the first eighteen aminoacids of such proteins, and conform to the sequence motifMet-Gly-Cys-Xaa-Cys, where the Met is cleaved, the Gly is N-acylated andone of the Cys residues is S-acylated. The Gly often is myristoylatedand a Cys can be palmitoylated. Acylation regions conforming to thesequence motif Cys-Ala-Ala-Xaa (so called “CAAX boxes”), which canmodified with C15 or C10 isoprenyl moieties, from the C-terminus ofG-protein gamma subunits and other proteins (e.g., World Wide Webaddress ebi.ac.uk/interpro/DisplaylproEntry?ac=IPR001230) also can beutilized. These and other acylation motifs include, for example, thosediscussed in Gauthier-Campbell et al., Molecular Biology of the Cell 15:2205-2217 (2004); Glabati et al., Biochem. J. 303: 697-700 (1994) andZlakine et al., J. Cell Science 110: 673-679 (1997), and can beincorporated in chimeric molecules to induce membrane localization. Incertain embodiments, a native sequence from a protein containing anacylation motif is incorporated into a chimeric protein. For example, insome embodiments, an N-terminal portion of Lck, Fyn or Yes or aG-protein alpha subunit, such as the first twenty-five N-terminal aminoacids or fewer from such proteins (e.g., about 5 to about 20 aminoacids, about 10 to about 19 amino acids, or about 15 to about 19 aminoacids of the native sequence with optional mutations), may beincorporated within the N-terminus of a chimeric protein. In certainembodiments, a C-terminal sequence of about 25 amino acids or less froma G-protein gamma subunit containing a CAAX box motif sequence (e.g.,about 5 to about 20 amino acids, about 10 to about 18 amino acids, orabout 15 to about 18 amino acids of the native sequence with optionalmutations) can be linked to the C-terminus of a chimeric protein. Insome embodiments, an acyl moiety has a log p value of +1 to +6, andsometimes has a log p value of +3 to +4.5. Log p values are a measure ofhydrophobicity and often are derived from octanol/water partitioningstudies, in which molecules with higher hydrophobicity partition intooctanol with higher frequency and are characterized as having a higherlog p value. Log p values are published for a number of lipophilicmolecules and log p values can be calculated using known partitioningprocesses (e.g., Chemical Reviews, Vol. 71, Issue 6, page 599, whereentry 4493 shows lauric acid having a log p value of 4.2). Any acylmoiety can be linked to a peptide composition discussed above and testedfor antimicrobial activity using known methods and those discussedhereafter. The acyl moiety sometimes is a C1-C20 alkyl, C2-C20 alkenyl,C2-C20 alkynyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C4-C12cyclalkylalkyl, aryl, substituted aryl, or aryl (C1-C4) alkyl, forexample. Any acyl-containing moiety sometimes is a fatty acid, andexamples of fatty acid moieties are propyl (C3), butyl (C4), pentyl(C5), hexyl (C6), heptyl (C7), octyl (C8), nonyl (C9), decyl (C10),undecyl (C11), lauryl (C12), myristyl (C14), palmityl (C16), stearyl(C18), arachidyl (C20), behenyl (C22) and lignoceryl moieties (C24), andeach moiety can contain 0, 1, 2, 3, 4, 5, 6, 7 or 8 unsaturations (i.e.,double bonds). An acyl moiety sometimes is a lipid molecule, such as aphosphatidyl lipid (e.g., phosphatidyl serine, phosphatidyl inositol,phosphatidyl ethanolamine, phosphatidyl choline), sphingolipid (e.g.,shingomyelin, sphingosine, ceramide, ganglioside, cerebroside), ormodified versions thereof. In certain embodiments, one, two, three, fouror five or more acyl moieties are linked to a membrane associationregion. Any membrane-targeting sequence can be employed that isfunctional in the host and may, or may not, be associated with one ofthe other domains of the chimeric protein. In some embodiments, suchsequences include, but are not limited to myristoylation-targetingsequence, palmitoylation-targeting sequence, prenylation sequences(i.e., farnesylation, geranyl-geranylation, CAAX Box), protein-proteininteraction motifs or transmembrane sequences (utilizing signalpeptides) from receptors. Examples include those discussed in, forexample, ten Klooster J P et al, Biology of the Cell (2007) 99, 1-12,Vincent, S., et al., Nature Biotechnology 21:936-40, 1098 (2003).

Additional protein domains exist that can increase protein retention atvarious membranes. For example, an ˜120 amino acid pleckstrin homology(PH) domain is found in over 200 human proteins that are typicallyinvolved in intracellular signaling. PH domains can bind variousphosphatidylinositol (PI) lipids within membranes (e.g. PI (3, 4,5)-P3,PI (3,4)-P2, PI (4,5)-P2) and thus play a key role in recruitingproteins to different membrane or cellular compartments. Often thephosphorylation state of PI lipids is regulated, such as by PI-3 kinaseor PTEN, and thus, interaction of membranes with PH domains are not asstable as by acyl lipids.

Where a polypeptide does not include a membrane-targeting region, suchas certain chimeric stimulating molecules provided herein, thepolypeptide does not include a region that provides for transport of thechimeric protein to a cell membrane. Where a polypeptide comprising amembrane-targeting region may be targeted to a membrane and localized toa 2-dimensional surface of the cell, a polypeptide that does notcomprise a membrane-targeting-region or a functional membrane-targetingregion will be non-localized in the cytosol. The polypeptide may, forexample, not include a sequence that transports the polypeptide to thecell surface membrane, or the polypeptide may, for example, include adysfunctional membrane-targeting region, that does not transport thepolypeptide to the cell surface membrane, for example, a myristoylationregion that includes a proline that disrupts the function of themyristoylation-targeting region. (see, for example, Resh, M. D.,Biochim. Biophys. Acta. 1451:1-16 (1999)). Polypeptides that are nottransported to the membrane are considered to be cytoplasmicpolypeptides, such as, for example, the cytoplasmic chimeric stimulatingmolecules discussed herein. Such cytoplasmic chimeric stimulatingmolecules may lack a membrane-targeting region, for example, or may lacka functional membrane-targeting region. By “cytoplasmic chimericstimulating molecule” is meant a polypeptide, such as the MyD88/CD40polypeptides discussed herein, that does not comprise an amino acidsequence that transports the polypeptide to the cell surface membrane,or includes a dysfunctional membrane-targeting region. A cytoplasmicchimeric stimulating molecule, or a polypeptide that comprises acytoplasmic chimeric stimulating molecule does not comprise an aminoacid sequence, or modified amino acid sequence, that is responsible fordirectly attaching the polypeptide to a lipid that associates with alipid membrane; a cytoplasmic chimeric stimulating molecule does notdirectly interact with lipids of the membrane. Thus, the term“cytoplasmic chimeric stimulating molecule” is not meant to includechimeric stimulating molecules that are part of a CAR polypeptidesequence, or other membrane-bound polypeptide. Following fluorescent orother labeling of a cell comprising a cytoplasmic chimeric stimulatingmolecule, the cytoplasmic stimulating molecule would be present in thecytoplasm of the cell, and would not stably touch, or directly interactfor a prolonged period with, the cytoplasmic hydrophobic lipid portionof the cell membrane.

Transmembrane Regions

A chimeric protein herein may include a single-pass or multiple passtransmembrane sequence (e.g., at the N-terminus or C-terminus of thechimeric protein). Single pass transmembrane regions are found incertain CD molecules, tyrosine kinase receptors, serine/threonine kinasereceptors, TGFβ, BMP, activin and phosphatases. Single passtransmembrane regions often include a signal peptide region and atransmembrane region of about 20 to about 25 amino acids, many of whichare hydrophobic amino acids and can form an alpha helix. A short trackof positively charged amino acids often follows the transmembrane spanto anchor the protein in the membrane. Multiple pass proteins includeion pumps, ion channels, and transporters, and include two or morehelices that span the membrane multiple times. All or substantially allof a multiple pass protein sometimes is incorporated in a chimericprotein. Sequences for single pass and multiple pass transmembraneregions are known and can be selected for incorporation into a chimericprotein molecule.

In some embodiments, the transmembrane domain is fused to theextracellular domain of the CAR. In one embodiment, the transmembranedomain that naturally is associated with one of the domains in the CARis used. In other embodiments, a transmembrane domain that is notnaturally associated with one of the domains in the CAR is used. In someinstances, the transmembrane domain can be selected or modified by aminoacid substitution (e.g., typically charged to a hydrophobic residue) toavoid binding of such domains to the transmembrane domains of the sameor different surface membrane proteins to minimize interactions withother members of the receptor complex.

Transmembrane domains may, for example, be derived from the alpha, beta,or zeta chain of the T cell receptor, CD3-ε, CD3ζ, CD4, CD5, CD8, CD8a,CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, orCD154. Or, in some examples, the transmembrane domain may be synthesizedde novo, comprising mostly hydrophobic residues, such as, for example,leucine and valine. In certain embodiments a short polypeptide linkermay form the linkage between the transmembrane domain and theintracellular domain of the chimeric antigen receptor. The chimericantigen receptors may further comprise a stalk, that is, anextracellular region of amino acids between the extracellular domain andthe transmembrane domain. For example, the stalk may be a sequence ofamino acids naturally associated with the selected transmembrane domain.In some embodiments, the chimeric antigen receptor comprises a CD8transmembrane domain, in certain embodiments, the chimeric antigenreceptor comprises a CD8 transmembrane domain, and additional aminoacids on the extracellular portion of the transmembrane domain, incertain embodiments, the chimeric antigen receptor comprises a CD8transmembrane domain and a CD8 stalk. The chimeric antigen receptor mayfurther comprise a region of amino acids between the transmembranedomain and the cytoplasmic domain, which are naturally associated withthe polypeptide from which the transmembrane domain is derived.

Control Regions

1. Promoters

The particular promoter employed to control the expression of apolynucleotide sequence of interest is not believed to be important, solong as it is capable of directing the expression of the polynucleotidein the targeted cell. Thus, where a human cell is targeted thepolynucleotide sequence-coding region may, for example, be placedadjacent to and under the control of a promoter that is capable of beingexpressed in a human cell. Generally speaking, such a promoter mightinclude either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, R-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it is desirable to prohibit orreduce expression of one or more of the transgenes. Examples oftransgenes that are toxic to the producer cell line are pro-apoptoticand cytokine genes. Several inducible promoter systems are available forproduction of viral vectors where the transgene products are toxic (addin more inducible promoters).

The ecdysone system (Life Technologies (formerly Invitrogen), Carlsbad,Calif.) is one such system. This system is designed to allow regulatedexpression of a gene of interest in mammalian cells. It consists of atightly regulated expression mechanism that allows virtually no basallevel expression of the transgene, but over 200-fold inducibility. Thesystem is based on the heterodimeric ecdysone receptor of Drosophila,and when ecdysone or an analog such as muristerone A binds to thereceptor, the receptor activates a promoter to turn on expression of thedownstream transgene high levels of mRNA transcripts are attained. Inthis system, both monomers of the heterodimeric receptor areconstitutively expressed from one vector, whereas theecdysone-responsive promoter, which drives expression of the gene ofinterest, is on another plasmid. Engineering of this type of system intothe gene transfer vector of interest would therefore be useful.Cotransfection of plasmids containing the gene of interest and thereceptor monomers in the producer cell line would then allow for theproduction of the gene transfer vector without expression of apotentially toxic transgene. At the appropriate time, expression of thetransgene could be activated with ecdysone or muristeron A.

Another inducible system that may be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-5551,1992; Gossen et al., Science, 268:1766-1769, 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstitutively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemmay be used so that the producer cells could be grown in the presence oftetracycline or doxycycline and prevent expression of a potentiallytoxic transgene, but when the vector is introduced to the patient, thegene expression would be constitutively on.

In some circumstances, it is desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity are utilized depending onthe level of expression desired. In mammalian cells, the CMV immediateearly promoter is often used to provide strong transcriptionalactivation. The CMV promoter is reviewed in Donnelly, J. J., et al.,1997. Annu. Rev. Immunol. 15:617-48. Modified versions of the CMVpromoter that are less potent have also been used when reduced levels ofexpression of the transgene are desired. When expression of a transgenein hematopoietic cells is desired, retroviral promoters such as the LTRsfrom MLV or MMTV are often used. Other viral promoters that are useddepending on the desired effect include SV40, RSV LTR, HIV-1 and HIV-2LTR, adenovirus promoters such as from the E1A, E2A, or MLP region, AAVLTR, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters are used to effect transcription inspecific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. These promoters may resultin reduced expression compared to a stronger promoter such as the CMVpromoter, but may also result in more limited expression, andimmunogenicity. (Bojak, A., et al., 2002. Vaccine. 20:1975-79; Cazeaux,N., et al., 2002. Vaccine 20:3322-31). For example, tissue specificpromoters such as the PSA associated promoter or prostate-specificglandular kallikrein, or the muscle creatine kinase gene may be usedwhere appropriate.

Examples of tissue specific or differentiation specific promotersinclude, but are not limited to, the following: B29/CD79b (B cells);CD14 (monocytic cells); CD43 (leukocytes and platelets); CD45(hematopoietic cells); CD68 (macrophages); desmin (muscle); elastase-1(pancreatic acinar cells); endoglin (endothelial cells); fibronectin(differentiating cells, healing tissues); and Flt-1 (endothelial cells);GFAP (astrocytes).

In certain indications, it is desirable to activate transcription atspecific times after administration of the gene therapy vector. This isdone with such promoters as those that are hormone or cytokineregulatable. Cytokine and inflammatory protein responsive promoters thatcan be used include K and T kininogen (Kageyama et al., (1987) J. Biol.Chem., 262, 2345-2351), c-fos, TNF-α, C-reactive protein (Arcone, etal., (1988) Nucl. Acids Res., 16(8), 3195-3207), haptoglobin (Olivieroet al., (1987) EMBO J., 6, 1905-1912), serum amyloid A2, C/EBP α, IL-1,IL-6 (Poli and Cortese, (1989) Proc. Nat'l Acad. Sci. USA, 86,8202-8206), Complement C3 (Wilson et al., (1990) Mol. Cell. Biol.,6181-6191), IL-8, α-1 acid glycoprotein (Prowse and Baumann, (1988) MolCell Biol, 8, 42-51), α-1 antitrypsin, lipoprotein lipase (Zechner etal., Mol. Cell. Biol., 2394-2401, 1988), angiotensinogen (Ron, et al.,(1991) Mol. Cell. Biol., 2887-2895), fibrinogen, c-jun (inducible byphorbol esters, TNF-α, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), α-2 macroglobulinand α-1 anti-chymotrypsin. Other promoters include, for example, SV40,MMTV, Human Immunodeficiency Virus (MV), Moloney virus, ALV, EpsteinBarr virus, Rous Sarcoma virus, human actin, myosin, hemoglobin, andcreatine.

It is envisioned that any of the above promoters alone or in combinationwith another can be useful depending on the action desired. Promoters,and other regulatory elements, are selected such that they arefunctional in the desired cells or tissue. In addition, this list ofpromoters should not be construed to be exhaustive or limiting; otherpromoters that are used in conjunction with the promoters and methodsdisclosed herein.

2. Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Early examples include the enhancers associated with immunoglobulin andT cell receptors that both flank the coding sequence and occur withinseveral introns. Many viral promoters, such as CMV, SV40, and retroviralLTRs are closely associated with enhancer activity and are often treatedlike single elements. Enhancers are organized much like promoters. Thatis, they are composed of many individual elements, each of which bindsto one or more transcriptional proteins. The basic distinction betweenenhancers and promoters is operational. An enhancer region as a wholestimulates transcription at a distance and often independent oforientation; this need not be true of a promoter region or its componentelements. On the other hand, a promoter has one or more elements thatdirect initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization. A subset ofenhancers includes locus-control regions (LCRs) that can not onlyincrease transcriptional activity, but (along with insulator elements)can also help to insulate the transcriptional element from adjacentsequences when integrated into the genome. Any promoter/enhancercombination (as per the Eukaryotic Promoter Data Base EPDB) can be usedto drive expression of the gene, although many will restrict expressionto a particular tissue type or subset of tissues. (Reviewed in, forexample, Kutzler, M. A., and Weiner, D. B., 2008. Nature ReviewsGenetics 9:776-88). Examples include, but are not limited to, enhancersfrom the human actin, myosin, hemoglobin, muscle creatine kinase,sequences, and from viruses CMV, RSV, and EBV. Appropriate enhancers maybe selected for particular applications. Eukaryotic cells can supportcytoplasmic transcription from certain bacterial promoters if theappropriate bacterial polymerase is provided, either as part of thedelivery complex or as an additional genetic expression construct.

3. Polyadenylation Signals

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the present methods, and anysuch sequence is employed such as human or bovine growth hormone andSV40 polyadenylation signals and LTR polyadenylation signals. Onenon-limiting example is the SV40 polyadenylation signal present in thepCEP3 plasmid (Invitrogen, Carlsbad, Calif.). Also contemplated as anelement of the expression cassette is a terminator. These elements canserve to enhance message levels and to minimize read through from thecassette into other sequences. Termination or poly(A) signal sequencesmay be, for example, positioned about 11-30 nucleotides downstream froma conserved sequence (AAUAAA) at the 3′ end of the mRNA. (Montgomery, D.L., et al., 1993. DNA Cell Biol. 12:777-83; Kutzler, M. A., and Weiner,D. B., 2008. Nature Rev. Gen. 9:776-88).

4. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.The initiation codon is placed in-frame with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments, the use of internal ribosome entry sites (IRES)elements is used to create multigene, or polycistronic messages. IRESelements are able to bypass the ribosome-scanning model of 5′ methylatedcap-dependent translation and begin translation at internal sites(Pelletier and Sonenberg, Nature, 334:320-325, 1988). IRES elements fromtwo members of the picornavirus family (polio and encephalomyocarditis)have been discussed (Pelletier and Sonenberg, 1988), as well an IRESfrom a mammalian message (Macejak and Sarnow, Nature, 353:90-94, 1991).IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

Sequence Optimization

Protein production may also be increased by optimizing the codons in thetransgene. Species specific codon changes may be used to increaseprotein production. Also, codons may be optimized to produce anoptimized RNA, which may result in more efficient translation. Byoptimizing the codons to be incorporated in the RNA, elements such asthose that result in a secondary structure that causes instability,secondary mRNA structures that can, for example, inhibit ribosomalbinding, or cryptic sequences that can inhibit nuclear export of mRNAcan be removed. (Kutzler, M. A., and Weiner, D. B., 2008. Nature Rev.Gen. 9:776-88; Yan, J. et al., 2007. Mol. Ther. 15:411-21; Cheung, Y.K., et al., 2004. Vaccine 23:629-38; Narum, D. L., et al., 2001.69:7250-55; Yadava, A., and Ockenhouse, C. F., 2003. Infect. Immun.71:4962-69; Smith, J. M., et al., 2004. AIDS Res. Hum. Retroviruses20:1335-47; Zhou, W., et al., 2002. Vet. Microbiol. 88:127-51; Wu, X.,et al., 2004. Biochem. Biophys. Res. Commun. 313:89-96; Zhang, W., etal., 2006. Biochem. Biophys. Res. Commun. 349:69-78; Deml, L. A., etal., 2001. J. Virol. 75:1099-11001; Schneider, R. M., et al., 1997. J.Virol. 71:4892-4903; Wang, S. D., et al., 2006. Vaccine 24:4531-40; zurMegede, J., et al., 2000. J. Virol. 74:2628-2635). For example, theFBP12 or other multimerizing region polypeptide, the co-stimulatingpolypeptide cytoplasmic signaling region, and the CD19 sequences may beoptimized by changes in the codons.

Leader Sequences

Leader sequences may be added to enhance the stability of mRNA andresult in more efficient translation. The leader sequence is usuallyinvolved in targeting the mRNA to the endoplasmic reticulum. Examplesinclude the signal sequence for the HIV-1 envelope glycoprotein (Env),which delays its own cleavage, and the IgE gene leader sequence(Kutzler, M. A., and Weiner, D. B., 2008. Nature Rev. Gen. 9:776-88; Li,V., et al., 2000. Virology 272:417-28; Xu, Z. L., et al. 2001. Gene272:149-56; Malin, A. S., et al., 2000. Microbes Infect. 2:1677-85;Kutzler, M. A., et al., 2005. J. Immunol. 175:112-125; Yang, J. S., etal., 2002. Emerg. Infect. Dis. 8:1379-84; Kumar, S., et al., 2006. DNACell Biol. 25:383-92; Wang, S., et al, 2006. Vaccine 24:4531-40). TheIgE leader may be used to enhance insertion into the endoplasmicreticulum (Tepler, I, et al. (1989) J. Biol. Chem. 264:5912).

Expression of the transgenes may be optimized and/or controlled by theselection of appropriate methods for optimizing expression. Thesemethods include, for example, optimizing promoters, delivery methods,and gene sequences, (for example, as presented in Laddy, D. J., et al.,2008. PLoS.ONE 3 e2517; Kutzler, M. A., and Weiner, D. B., 2008. NatureRev. Gen. 9:776-88).

Nucleic Acids

A “nucleic acid” as used herein generally refers to a molecule (one, twoor more strands) of DNA, RNA or a derivative or analog thereof,comprising a nucleobase. A nucleobase includes, for example, a naturallyoccurring purine or pyrimidine base found in DNA (e.g., an adenine “A,”a guanine “G,” a thymine “T” or a cytosine “C”) or RNA (e.g., an A, a G,an uracil “U” or a C). The term “nucleic acid” encompasses the terms“oligonucleotide” and “polynucleotide,” each as a subgenus of the term“nucleic acid.” Nucleic acids may be, be at least, be at most, or beabout 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500,510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920,930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides, or any rangederivable therein, in length.

Nucleic acids herein provided may have regions of identity orcomplementarity to another nucleic acid. It is contemplated that theregion of complementarity or identity can be at least 5 contiguousresidues, though it is specifically contemplated that the region is, isat least, is at most, or is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830,840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,980, 990, or 1000 contiguous nucleotides.

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean forming a double or triple strandedmolecule or a molecule with partial double or triple stranded nature.The term “anneal” as used herein is synonymous with “hybridize.” Theterm “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butpreclude hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are known, and are often used for applicationsrequiring high selectivity. Non-limiting applications include isolatinga nucleic acid, such as a gene or a nucleic acid segment thereof, ordetecting at least one specific mRNA transcript or a nucleic acidsegment thereof, and the like.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.5 M NaCl attemperatures of about 42 degrees C. to about 70 degrees C. It isunderstood that the temperature and ionic strength of a desiredstringency are determined in part by the length of the particularnucleic acid(s), the length and nucleobase content of the targetsequence(s), the charge composition of the nucleic acid(s), and thepresence or concentration of formamide, tetramethylammonium chloride orother solvent(s) in a hybridization mixture.

It is understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned varyingconditions of hybridization may be employed to achieve varying degreesof selectivity of a nucleic acid towards a target sequence. In anon-limiting example, identification or isolation of a related targetnucleic acid that does not hybridize to a nucleic acid under stringentconditions may be achieved by hybridization at low temperature and/orhigh ionic strength. Such conditions are termed “low stringency” or “lowstringency conditions,” and non-limiting examples of low stringencyinclude hybridization performed at about 0.15 M to about 0.9 M NaCl at atemperature range of about 20 degrees C. to about 50 degrees C. The lowor high stringency conditions may be further modified to suit aparticular application.

“Function-conservative variants” are proteins or enzymes in which agiven amino acid residue has been changed without altering overallconformation and function of the protein or enzyme, including, but notlimited to, replacement of an amino acid with one having similarproperties, including polar or non-polar character, size, shape andcharge. Conservative amino acid substitutions for many of the commonlyknown non-genetically encoded amino acids are well known in the art.Conservative substitutions for other non-encoded amino acids can bedetermined based on their physical properties as compared to theproperties of the genetically encoded amino acids.

Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and canbe, for example, at least 70%, for example, at least 80%, for example,at least 90%, and for example, at least 95%, as determined according toan alignment scheme. As referred to herein, “sequence similarity” meansthe extent to which nucleotide or protein sequences are related. Theextent of similarity between two sequences can be based on percentsequence identity and/or conservation. “Sequence identity” herein meansthe extent to which two nucleotide or amino acid sequences areinvariant. “Sequence alignment” means the process of lining up two ormore sequences to achieve maximal levels of identity (and, in the caseof amino acid sequences, conservation) for the purpose of assessing thedegree of similarity. Numerous methods for aligning sequences andassessing similarity/identity are known in the art such as, for example,the Cluster Method, wherein similarity is based on the MEGALIGNalgorithm, as well as BLASTN, BLASTP, and FASTA. When using any of theseprograms, in certain embodiments, the settings are those that results inthe highest sequence similarity.

Nucleic Acid Modification

Any of the modifications discussed below may be applied to a nucleicacid. Examples of modifications include alterations to the RNA or DNAbackbone, sugar or base, and various combinations thereof. Any suitablenumber of backbone linkages, sugars and/or bases in a nucleic acid canbe modified (e.g., independently about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, up to 100%).An unmodified nucleoside is any one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon of13-D-ribo-furanose.

A modified base is a nucleotide base other than adenine, guanine,cytosine and uracil at a 1′ position. Non-limiting examples of modifiedbases include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil,dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e. g.,5-methylcytidine), 5-alkyluridines (e. g., ribothymidine), 5-halouridine(e. g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e. g.6-methyluridine), propyne, and the like. Other non-limiting examples ofmodified bases include nitropyrrolyl (e.g., 3-nitropyrrolyl),nitroindolyl (e.g., 4-, 5-, 6-nitroindolyl), hypoxanthinyl, isoinosinyl,2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl,nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole,3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl,3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl,6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl,pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl,propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl,4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl,pyrenyl, stilbenyl, tetracenyl, pentacenyl and the like.

In some embodiments, for example, a nucleic acid may comprise modifiednucleic acid molecules, with phosphate backbone modifications.Non-limiting examples of backbone modifications includephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl modifications. In certain instances, aribose sugar moiety that naturally occurs in a nucleoside is replacedwith a hexose sugar, polycyclic heteroalkyl ring, or cyclohexenyl group.In certain instances, the hexose sugar is an allose, altrose, glucose,mannose, gulose, idose, galactose, talose, or a derivative thereof. Thehexose may be a D-hexose, glucose, or mannose. In certain instances, thepolycyclic heteroalkyl group may be a bicyclic ring containing oneoxygen atom in the ring. In certain instances, the polycyclicheteroalkyl group is a bicyclo[2.2.1]heptane, a bicyclo[3.2.1]octane, ora bicyclo[3.3.1]nonane.

Nitropyrrolyl and nitroindolyl nucleobases are members of a class ofcompounds known as universal bases. Universal bases are those compoundsthat can replace any of the four naturally occurring bases withoutsubstantially affecting the melting behavior or activity of theoligonucleotide duplex. In contrast to the stabilizing, hydrogen-bondinginteractions associated with naturally occurring nucleobases,oligonucleotide duplexes containing 3-nitropyrrolyl nucleobases may bestabilized solely by stacking interactions. The absence of significanthydrogen-bonding interactions with nitropyrrolyl nucleobases obviatesthe specificity for a specific complementary base. In addition, 4-, 5-and 6-nitroindolyl display very little specificity for the four naturalbases. Procedures for the preparation of1-(2′-O-methyl-.β.-D-ribofuranosyl)-5-nitroindole are discussed inGaubert, G.; Wengel, J. Tetrahedron Letters 2004, 45, 5629. Otheruniversal bases include hypoxanthinyl, isoinosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, and structuralderivatives thereof.

Difluorotolyl is a non-natural nucleobase that functions as a universalbase. Difluorotolyl is an isostere of the natural nucleobase thymine.But unlike thymine, difluorotolyl shows no appreciable selectivity forany of the natural bases. Other aromatic compounds that function asuniversal bases are 4-fluoro-6-methylbenzimidazole and4-methylbenzimidazole. In addition, the relatively hydrophobicisocarbostyrilyl derivatives 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl are universalbases which cause only slight destabilization of oligonucleotideduplexes compared to the oligonucleotide sequence containing onlynatural bases. Other non-natural nucleobases include 7-azaindolyl,6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl,pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl,propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl,4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl,pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatesthereof. For a more detailed discussion, including synthetic procedures,of difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole,and other non-natural bases mentioned above, see: Schweitzer et al., J.Org. Chem., 59:7238-7242 (1994);

In addition, chemical substituents, for example cross-linking agents,may be used to add further stability or irreversibility to the reaction.Non-limiting examples of cross-linking agents include, for example,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl) dithio]propioimidate.

A nucleotide analog may also include a “locked” nucleic acid. Certaincompositions can be used to essentially “anchor” or “lock” an endogenousnucleic acid into a particular structure. Anchoring sequences serve toprevent disassociation of a nucleic acid complex, and thus not only canprevent copying but may also enable labeling, modification, and/orcloning of the endogenous sequence. The locked structure may regulategene expression (i.e. inhibit or enhance transcription or replication),or can be used as a stable structure that can be used to label orotherwise modify the endogenous nucleic acid sequence, or can be used toisolate the endogenous sequence, i.e. for cloning.

Nucleic acid molecules need not be limited to those molecules containingonly RNA or DNA, but further encompass chemically modified nucleotidesand non-nucleotides. The percent of non-nucleotides or modifiednucleotides may be from 1% to 100% (e.g., about 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%).

Nucleic Acid Preparation

In some embodiments, a nucleic acid is provided for use as a control orstandard in an assay, or therapeutic, for example. A nucleic acid may bemade by any technique known in the art, such as for example, chemicalsynthesis, enzymatic production or biological production. Nucleic acidsmay be recovered or isolated from a biological sample. The nucleic acidmay be recombinant or it may be natural or endogenous to the cell(produced from the cell's genome). It is contemplated that a biologicalsample may be treated in a way so as to enhance the recovery of smallnucleic acid molecules. Generally, methods may involve lysing cells witha solution having guanidinium and a detergent.

Nucleic acid synthesis may also be performed according to standardmethods. Non-limiting examples of a synthetic nucleic acid (e.g., asynthetic oligonucleotide), include a nucleic acid made by in vitrochemical synthesis using phosphotriester, phosphite, or phosphoramiditechemistry and solid phase techniques or via deoxynucleosideH-phosphonate intermediates. Various different mechanisms ofoligonucleotide synthesis have been disclosed elsewhere.

Nucleic acids may be isolated using known techniques. In particularembodiments, methods for isolating small nucleic acid molecules, and/orisolating RNA molecules can be employed. Chromatography is a processused to separate or isolate nucleic acids from protein or from othernucleic acids. Such methods can involve electrophoresis with a gelmatrix, filter columns, alcohol precipitation, and/or otherchromatography. If a nucleic acid from cells is to be used or evaluated,methods generally involve lysing the cells with a chaotropic (e.g.,guanidinium isothiocyanate) and/or detergent (e.g., N-lauroyl sarcosine)prior to implementing processes for isolating particular populations ofRNA.

Methods may involve the use of organic solvents and/or alcohol toisolate nucleic acids. In some embodiments, the amount of alcohol addedto a cell lysate achieves an alcohol concentration of about 55% to 60%.While different alcohols can be employed, ethanol works well. A solidsupport may be any structure, and it includes beads, filters, andcolumns, which may include a mineral or polymer support withelectronegative groups. A glass fiber filter or column is effective forsuch isolation procedures.

A nucleic acid isolation processes may sometimes include: a) lysingcells in the sample with a lysing solution comprising guanidinium, wherea lysate with a concentration of at least about 1 M guanidinium isproduced; b) extracting nucleic acid molecules from the lysate with anextraction solution comprising phenol; c) adding to the lysate analcohol solution for form a lysate/alcohol mixture, wherein theconcentration of alcohol in the mixture is between about 35% to about70%; d) applying the lysate/alcohol mixture to a solid support; e)eluting the nucleic acid molecules from the solid support with an ionicsolution; and, f) capturing the nucleic acid molecules. The sample maybe dried down and resuspended in a liquid and volume appropriate forsubsequent manipulation.

Methods of Gene Transfer

In order to mediate the effect of the transgene expression in a cell, itwill be necessary to transfer the expression constructs into a cell.Such transfer may employ viral or non-viral methods of gene transfer.This section provides a discussion of methods and compositions of genetransfer. A transformed cell comprising an expression vector isgenerated by introducing into the cell the expression vector. Suitablemethods for polynucleotide delivery for transformation of an organelle,a cell, a tissue or an organism for use with the current methods includevirtually any method by which a polynucleotide (e.g., DNA) can beintroduced into an organelle, a cell, a tissue or an organism.

A host cell can, and has been, used as a recipient for vectors. Hostcells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded polynucleotide sequences. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained through the American Type Culture Collection (ATCC),which is an organization that serves as an archive for living culturesand genetic materials. In specific embodiments, the host cell is a Tcell, a tumor-infiltrating lymphocyte, a natural killer cell, or anatural killer T cell.

An appropriate host may be determined. Generally this is based on thevector backbone and the desired result. A plasmid or cosmid, forexample, can be introduced into a prokaryote host cell for replicationof many vectors. Bacterial cells used as host cells for vectorreplication and/or expression include DH5a, JM109, and KCB, as well as anumber of commercially available bacterial hosts such as SURE® CompetentCells and SOLOPACK Gold Cells (STRATAGENE®, La Jolla, Calif.).Alternatively, bacterial cells such as E. coli LE392 could be used ashost cells for phage viruses. Eukaryotic cells that can be used as hostcells include, but are not limited to yeast, insects and mammals.Examples of mammalian eukaryotic host cells for replication and/orexpression of a vector include, but are not limited to, HeLa, NIH3T3,Jurkat, 293, COS, CHO, Saos, and PC12. Examples of yeast strainsinclude, but are not limited to, YPH499, YPH500 and YPH501.

Nucleic acid vaccines may include, for example, non-viral DNA vectors,“naked” DNA and RNA, and viral vectors. Methods of transforming cellswith these vaccines, and for optimizing the expression of genes includedin these vaccines are known and are also discussed herein.

Examples of Methods of Nucleic Acid or Viral Vector Transfer

Any appropriate method may be used to transfect or transform the cells,for example, the T cells, or to administer the nucleotide sequences orcompositions of the present methods. Certain examples are presentedherein, and further include methods such as delivery using cationicpolymers, lipid like molecules, and certain commercial products such as,for example, IN-VIVO-JET PEI.

1. Ex vivo Transformation

Various methods are available for transfecting vascular cells andtissues removed from an organism in an ex vivo setting. For example,canine endothelial cells have been genetically altered by retroviralgene transfer in vitro and transplanted into a canine (Wilson et al.,Science, 244:1344-1346, 1989). In another example, Yucatan minipigendothelial cells were transfected by retrovirus in vitro andtransplanted into an artery using a double-balloon catheter (Nabel etal., Science, 244(4910):1342-1344, 1989). Thus, it is contemplated thatcells or tissues may be removed and transfected ex vivo using thepolynucleotides presented herein. In particular aspects, thetransplanted cells or tissues may be placed into an organism. Forexample, T cells may be obtained from an animal, the cells transfectedor transduced with the expression vector and then administered back tothe animal.

2. Injection

In certain embodiments, a cell or a nucleic acid or viral vector may bedelivered to an organelle, a cell, a tissue or an organism via one ormore injections (i.e., a needle injection), such as, for example,subcutaneous, intradermal, intramuscular, intravenous, intraprotatic,intratumor, intraperitoneal, etc. Methods of injection include, foeexample, injection of a composition comprising a saline solution.Further embodiments include the introduction of a polynucleotide bydirect microinjection. The amount of the expression vector used may varyupon the nature of the antigen as well as the organelle, cell, tissue ororganism used.

Intradermal, intranodal, or intralymphatic injections are some of themore commonly used methods of DC administration. Intradermal injectionis characterized by a low rate of absorption into the bloodstream butrapid uptake into the lymphatic system. The presence of large numbers ofLangerhans dendritic cells in the dermis will transport intact as wellas processed antigen to draining lymph nodes. Proper site preparation isnecessary to perform this correctly (i.e., hair is clipped in order toobserve proper needle placement). Intranodal injection allows for directdelivery of antigen to lymphoid tissues. Intralymphatic injection allowsdirect administration of DCs.

3. Electroporation

In certain embodiments, a polynucleotide is introduced into anorganelle, a cell, a tissue or an organism via electroporation.Electroporation involves the exposure of a suspension of cells and DNAto a high-voltage electric discharge. In some variants of this method,certain cell wall-degrading enzymes, such as pectin-degrading enzymes,are employed to render the target recipient cells more susceptible totransformation by electroporation than untreated cells (U.S. Pat. No.5,384,253, incorporated herein by reference).

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humanK-immunoglobulin genes (Potter et al., (1984) Proc. Nat'l Acad. Sci.USA, 81, 7161-7165), and rat hepatocytes have been transfected with thechloramphenicol acetyltransferase gene (Tur-Kaspa et al., (1986) Mol.Cell Biol., 6, 716-718) in this manner.

In vivo electroporation for vaccines, or eVac, is clinically implementedthrough a simple injection technique. A DNA vector encoding tumorantigen is injected intradermally in a patient. Then electrodes applyelectrical pulses to the intradermal space causing the cells localizedthere, especially resident dermal dendritic cells, to take up the DNAvector and express the encoded tumor antigen. These tumorantigen-expressing dendritic cells activated by local inflammation canthen migrate to lymph-nodes, presenting tumor antigens and priming tumorantigen-specific T cells. A nucleic acid is electroporeticallyadministered when it is administered using electroporation, following,for example, but not limited to, injection of the nucleic acid or anyother means of administration where the nucleic acid may be delivered tothe cells by electroporation

Methods of electroporation are discussed in, for example, Sardesai,N.Y., and Weiner, D. B., Current Opinion in Immunotherapy 23:421-9(2011) and Ferraro, B. et al., Human Vaccines 7:120-127 (2011), whichare hereby incorporated by reference herein in their entirety.

4. Calcium Phosphate

In other embodiments, a polynucleotide is introduced to the cells usingcalcium phosphate precipitation. Human KB cells have been transfectedwith adenovirus 5 DNA (Graham and van der Eb, (1973) Virology, 52,456-467) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752,1987), and rat hepatocytes were transfected with a variety of markergenes (Rippe et al., Mol. Cell Biol., 10:689-695, 1990).

5. DEAE-Dextran

In another embodiment, a polynucleotide is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, T. V., Mol Cell Biol. 1985 May; 5(5):1188-90).

6. Sonication Loading

Additional embodiments include the introduction of a polynucleotide bydirect sonic loading. LTK-fibroblasts have been transfected with thethymidine kinase gene by sonication loading (Fechheimer et al., (1987)Proc. Nat'l Acad. Sci. USA, 84, 8463-8467).

7. Liposome-Mediated Transfection

In a further embodiment, a polynucleotide may be entrapped in a lipidcomplex such as, for example, a liposome. Liposomes are vesicularstructures characterized by a phospholipid bilayer membrane and an inneraqueous medium. Multilamellar liposomes have multiple lipid layersseparated by aqueous medium. They form spontaneously when phospholipidsare suspended in an excess of aqueous solution. The lipid componentsundergo self-rearrangement before the formation of closed structures andentrap water and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and TherapyUsing Specific Receptors and Ligands. pp. 87-104). Also contemplated isa polynucleotide complexed with Lipofectamine (Gibco BRL) or Superfect(Qiagen).

Receptor-Mediated Transfection

Still further, a polynucleotide may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a polynucleotide-binding agent. Otherscomprise a cell receptor-specific ligand to which the polynucleotide tobe delivered has been operatively attached. Several ligands have beenused for receptor-mediated gene transfer (Wu and Wu, (1987) J. Biol.Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA,87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA,91:4086-4090, 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been discussed (Wu and Wu, Adv. DrugDelivery Rev., 12:159-167, 1993; incorporated herein by reference). Incertain aspects, a ligand is chosen to correspond to a receptorspecifically expressed on the target cell population. In otherembodiments, a polynucleotide delivery vehicle component of acell-specific polynucleotide-targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The polynucleotide(s) tobe delivered are housed within the liposome and the specific bindingligand is functionally incorporated into the liposome membrane. Theliposome will thus specifically bind to the receptor(s) of a target celland deliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a polynucleotide tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the polynucleotide delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichmay, for example, comprise one or more lipids or glycoproteins thatdirect cell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialoganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., (1987) Methods Enzymol., 149, 157-176). Itis contemplated that the tissue-specific transforming constructs may bespecifically delivered into a target cell in a similar manner.

8. Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce apolynucleotide into at least one, organelle, cell, tissue or organism(U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is incorporated herein by reference). Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., (1987) Nature, 327, 70-73).There are a wide variety of microprojectile bombardment techniques knownin the art, many of which are applicable to the present methods.

In this microprojectile bombardment, one or more particles may be coatedwith at least one polynucleotide and delivered into cells by apropelling force. Several devices for accelerating small particles havebeen developed. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568-9572). Themicroprojectiles used have consisted of biologically inert substancessuch as tungsten or gold particles or beads. Exemplary particles includethose comprised of tungsten, platinum, and, in certain examples, gold,including, for example, nanoparticles. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

Examples of Methods of Viral Vector-Mediated Transfer

Any viral vector suitable for administering nucleotide sequences, orcompositions comprising nucleotide sequences, to a cell or to a subject,such that the cell or cells in the subject may express the genes encodedby the nucleotide sequences may be employed in the present methods. Incertain embodiments, a transgene is incorporated into a viral particleto mediate gene transfer to a cell. Typically, the virus simply will beexposed to the appropriate host cell under physiologic conditions,permitting uptake of the virus. The present methods are advantageouslyemployed using a variety of viral vectors, as discussed below.

Adenovirus

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kb viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, M. J. (1990) Radiother Oncol., 19, 197-218). The products ofthe late genes (L1, L2, L3, L4 and L5), including the majority of theviral capsid proteins, are expressed only after significant processingof a single primary transcript issued by the major late promoter (MLP).The MLP (located at 16.8 map units) is particularly efficient during thelate phase of infection, and all the mRNAs issued from this promoterpossess a 5′ tripartite leader (TL) sequence, which makes them usefulfor translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous in that elimination ofadenoviral genes serves both ends. By practice of the present methods,it is possible to achieve both these goals while retaining the abilityto manipulate the therapeutic constructs with relative ease.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay, R. T., et al., J Mol Biol. 1984 Jun. 5;175(4):493-510). Therefore, inclusion of these elements in an adenoviralvector may permits replication.

In addition, the packaging signal for viral encapsulation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., J. (1987) Virol., 67, 2555-2558). This signalmimics the protein recognition site in bacteriophage lambda DNA where aspecific sequence close to the left end, but outside the cohesive endsequence, mediates the binding to proteins that are required forinsertion of the DNA into the head structure. E1 substitution vectors ofAd have demonstrated that a 450 bp (0-1.25 map units) fragment at theleft end of the viral genome could direct packaging in 293 cells(Levrero et al., Gene, 101:195-202, 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element derives from the packagingfunction of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts et. al. (1977)Cell, 12, 243-249). Later studies showed that a mutant with a deletionin the E1A (194-358 bp) region of the genome grew poorly even in a cellline that complemented the early (E1A) function (Hearing and Shenk,(1983) J. Mol. Biol. 167, 809-822). When a compensating adenoviral DNA(0-353 bp) was recombined into the right end of the mutant, the viruswas packaged normally. Further mutational analysis identified a short,repeated, position-dependent element in the left end of the Ad5 genome.One copy of the repeat was found to be sufficient for efficientpackaging if present at either end of the genome, but not when movedtoward the interior of the Ad5 DNA molecule (Hearing et al., J. (1987)Virol., 67, 2555-2558).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals is packagedselectively when compared to the helpers. If the preference is greatenough, stocks approaching homogeneity may be achieved.

To improve the tropism of ADV constructs for particular tissues orspecies, the receptor-binding fiber sequences can often be substitutedbetween adenoviral isolates. For example the Coxsackie-adenovirusreceptor (CAR) ligand found in adenovirus 5 can be substituted for theCD46-binding fiber sequence from adenovirus 35, making a virus withgreatly improved binding affinity for human hematopoietic cells. Theresulting “pseudotyped” virus, Ad5f35, has been the basis for severalclinically developed viral isolates. Moreover, various biochemicalmethods exist to modify the fiber to allow re-targeting of the virus totarget cells, such as, for example, T cells. Methods include use ofbifunctional antibodies (with one end binding the CAR ligand and one endbinding the target sequence), and metabolic biotinylation of the fiberto permit association with customized avidin-based chimeric ligands.Alternatively, one could attach ligands (e.g. anti-CD205 byheterobifunctional linkers (e.g. PEG-containing), to the adenovirusparticle.

1. Retrovirus

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, (1990)In: Virology, ed., New York: Raven Press, pp. 1437-1500). The resultingDNA then stably integrates into cellular chromosomes as a provirus anddirects synthesis of viral proteins. The integration results in theretention of the viral gene sequences in the recipient cell and itsdescendants. The retroviral genome contains three genes—gag, pol andenv—that code for capsid proteins, polymerase enzyme, and envelopecomponents, respectively. A sequence found upstream from the gag gene,termed psi, functions as a signal for packaging of the genome intovirions. Two long terminal repeat (LTR) sequences are present at the 5′and 3′ ends of the viral genome. These contain strong promoter andenhancer sequences and also are required for integration in the hostcell genome (Coffin, 1990). Thus, for example, the present technologyincludes, for example, cells whereby the polynucleotide used totransduce the cell is integrated into the genome of the cell.

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and psi components is constructed (Mann etal., (1983) Cell, 33, 153-159). When a recombinant plasmid containing ahuman cDNA, together with the retroviral LTR and psi sequences isintroduced into this cell line (by calcium phosphate precipitation forexample), the psi sequence allows the RNA transcript of the recombinantplasmid to be packaged into viral particles, which are then secretedinto the culture media (Nicolas, J. F., and Rubenstein, J. L. R., (1988)In: Vectors: a Survey of Molecular Cloning Vectors and Their Uses,Rodriquez and Denhardt, Eds.). Nicolas and Rubenstein; Temin et al.,(1986) In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press,pp. 149-188; Mann et al., 1983). The media containing the recombinantretroviruses is collected, optionally concentrated, and used for genetransfer. Retroviral vectors are able to infect a broad variety of celltypes. However, integration and stable expression of many types ofretroviruses require the division of host cells (Paskind et al., (1975)Virology, 67, 242-248). An approach designed to allow specific targetingof retrovirus vectors recently was developed based on the chemicalmodification of a retrovirus by the chemical addition of galactoseresidues to the viral envelope. This modification could permit thespecific infection of cells such as hepatocytes via asialoglycoproteinreceptors, may this be desired.

A different approach to targeting of recombinant retroviruses wasdesigned, which used biotinylated antibodies against a retroviralenvelope protein and against a specific cell receptor. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., (1989) Proc. Nat'l Acad. Sci. USA, 86, 9079-9083). Using antibodiesagainst major histocompatibility complex class I and class II antigens,the infection of a variety of human cells that bore those surfaceantigens was demonstrated with an ecotropic virus in vitro (Roux et al.,1989).

2. Adeno-Associated Virus

AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.Inverted terminal repeats flank the genome. Two genes are present withinthe genome, giving rise to a number of distinct gene products. Thefirst, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription. The three promotersin AAV are designated by their location, in map units, in the genome.These are, from left to right, p5, p19 and p40. Transcription gives riseto six transcripts, two initiated at each of three promoters, with oneof each pair being spliced. The splice site, derived from map units42-46, is the same for each transcript. The four non-structural proteinsapparently are derived from the longer of the transcripts, and threevirion proteins all arise from the smallest transcript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low-levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al., J. Virol., 61:3096-3101 (1987)),or by other methods, including but not limited to chemical or enzymaticsynthesis of the terminal repeats based upon the published sequence ofAAV. It can be determined, for example, by deletion analysis, theminimum sequence or part of the AAV ITRs which is required to allowfunction, i.e., stable and site-specific integration. It can also bedetermined which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,(1995) Ann. N.Y. Acad. Sci., 770; 79-90; Chatteijee, et al., (1995) Ann.N.Y. Acad. Sci., 770, 79-90; Ferrari et al., (1996) J. Virol., 70,3227-3234; Fisher et al., (1996) J. Virol., 70, 520-532; Flotte et al.,Proc. Nat'l Acad. Sci. USA, 90, 10613-10617, (1993); Goodman et al.(1994), Blood, 84, 1492-1500; Kaplitt et al., (1994) Nat'l Genet., 8,148-153; Kaplitt, M. G., et al., Ann Thorac Surg. 1996 December;62(6):1669-76; Kessler et al., (1996) Proc. Nat'l Acad. Sci. USA, 93,14082-14087; Koeberl et al., (1997) Proc. Nat'l Acad. Sci. USA, 94,1426-1431; Mizukami et al., (1996) Virology, 217, 124-130).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1995; Flotte et al., Proc. Nat'l Acad. Sci. USA, 90,10613-10617, (1993)). Similarly, the prospects for treatment of musculardystrophy by AAV-mediated gene delivery of the dystrophin gene toskeletal muscle, of Parkinson's disease by tyrosine hydroxylase genedelivery to the brain, of hemophilia B by Factor IX gene delivery to theliver, and potentially of myocardial infarction by vascular endothelialgrowth factor gene to the heart, appear promising since AAV-mediatedtransgene expression in these organs has recently been shown to behighly efficient (Fisher et al., (1996) J. Virol., 70, 520-532; Flotteet al., 1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCownet al., (1996) Brain Res., 713, 99-107; Ping et al., (1996)Microcirculation, 3, 225-228; Xiao et al., (1996) J. Virol., 70,8098-8108).

3. Other Viral Vectors

Other viral vectors are employed as expression constructs in the presentmethods and compositions. Vectors derived from viruses such as vacciniavirus (Ridgeway, (1988) In: Vectors: A survey of molecular cloningvectors and their uses, pp. 467-492; Baichwal and Sugden, (1986) In,Gene Transfer, pp. 117-148; Coupar et al., Gene, 68:1-10, 1988) canarypoxvirus, and herpes viruses are employed. These viruses offer severalfeatures for use in gene transfer into various mammalian cells.

Once the construct has been delivered into the cell, the nucleic acidencoding the transgene are positioned and expressed at different sites.In certain embodiments, the nucleic acid encoding the transgene isstably integrated into the genome of the cell. This integration is inthe cognate location and orientation via homologous recombination (genereplacement) or it is integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid isstably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

Methods for Treating a Disease

The present methods also encompass methods of treatment or prevention ofa disease where administration of cells by, for example, infusion, maybe beneficial.

Cells, such as, for example, T cells, tumor infiltrating lymphocytes,natural killer cells, natural killer T cells, or progenitor cells, suchas, for example, hematopoietic stem cells, mesenchymal stromal cells,stem cells, pluripotent stem cells, and embryonic stem cells may be usedfor cell therapy. The cells may be from a donor, or may be cellsobtained from the patient. The cells may, for example, be used inregeneration, for example, to replace the function of diseased cells.The cells may also be modified to express a heterologous gene so thatbiological agents may be delivered to specific microenvironments suchas, for example, diseased bone marrow or metastatic deposits.Mesenchymal stromal cells have also, for example, been used to provideimmunosuppressive activity, and may be used in the treatment of graftversus host disease and autoimmune disorders. The cells provided in thepresent application contain a safety switch that may be valuable in asituation where following cell therapy, the activity of the therapeuticcells needs to be increased, or decreased. For example, where T cellsthat express a chimeric antigen receptor are provided to the patient, insome situations there may be an adverse event, such as off-targettoxicity. Ceasing the administration of the ligand would return thetherapeutic T cells to a non-activated state, remaining at a low,non-toxic, level of expression. Or, for example, the therapeutic cellmay work to decrease the tumor cell, or tumor size, and may no longer beneeded. In this situation, administration of the ligand may cease, andthe therapeutic cells would no longer be activated. If the tumor cellsreturn, or the tumor size increases following the initial therapy, theligand may be administered again, in order to activate the chimericantigen receptor-expressing T cells, and re-treat the patient.

By “therapeutic cell” is meant a cell used for cell therapy, that is, acell administered to a subject to treat or prevent a condition ordisease.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of pharmaceutical compositioncalculated to produce the desired immune-stimulating effect inassociation with the required diluent. The specifications for the unitdose of an inoculum are dictated by and are dependent upon the uniquecharacteristics of the pharmaceutical composition and the particularimmunologic effect to be achieved.

An effective amount of the pharmaceutical composition, such as themultimeric ligand presented herein, would be the amount that achievesthis selected result of inducing apoptosis in the Caspase-9-expressingcells T cells, such that over 60%, 70%, 80%, 85%, 90%, 95%, or 97%, orthat under 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the therapeuticcells are killed. The term is also synonymous with “sufficient amount.”The effective amount where the pharmaceutical composition is themodified T cell may also be the amount that achieves the desiredtherapeutic response, such as, the reduction of tumor size, the decreasein the level of tumor cells, or the decrease in the level ofCD19-expressing leukemic cells, compared to the time before the ligandinducer is administered.

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One can empiricallydetermine the effective amount of a particular composition presentedherein without necessitating undue experimentation.

The terms “contacted” and “exposed,” when applied to a cell, tissue ororganism, are used herein to describe the process by which thepharmaceutical composition and/or another agent, such as for example achemotherapeutic or radiotherapeutic agent, are delivered to a targetcell, tissue or organism or are placed in direct juxtaposition with thetarget cell, tissue or organism. To achieve cell killing or stasis, thepharmaceutical composition and/or additional agent(s) are delivered toone or more cells in a combined amount effective to kill the cell(s) orprevent them from dividing.

The administration of the pharmaceutical composition may precede, beconcurrent with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the pharmaceuticalcomposition and other agent(s) are applied separately to a cell, tissueor organism, one would generally ensure that a significant period oftime did not expire between the times of each delivery, such that thepharmaceutical composition and agent(s) would still be able to exert anadvantageously combined effect on the cell, tissue or organism. Forexample, in such instances, it is contemplated that one may contact thecell, tissue or organism with two, three, four or more modalitiessubstantially simultaneously (i.e., within less than about a minute)with the pharmaceutical composition. In other aspects, one or moreagents may be administered within of from substantially simultaneously,about 1 minute, to about 24 hours to about 7 days to about 1 to about 8weeks or more, and any range derivable therein, prior to and/or afteradministering the expression vector. Yet further, various combinationregimens of the pharmaceutical composition presented herein and one ormore agents may be employed.

Optimized and Personalized Therapeutic Treatment

The dosage and administration schedule of the modified cells may beoptimized by determining the level of the disease or condition to betreated. For example, the size of any remaining solid tumor, or thelevel of targeted cells such as, for example, tumor cells orCD19-expressing B cells, which remain in the patient, may be determined.

For example, determining that a patient has clinically relevant levelsof tumor cells, or a solid tumor, after initial therapy, provides anindication to a clinician that it may be necessary to administer themodified T cells. In another example, determining that a patient has areduced level of tumor cells or reduced tumor size after treatment withthe modified cells may indicate to the clinician that no additional doseof the modified cells is needed. Similarly, after treatment with themodified cells, determining that the patient continues to exhibitdisease or condition symptoms, or suffers a relapse of symptoms mayindicate to the clinician that it may be necessary to administer atleast one additional dose of modified cells.

The term “dosage” is meant to include both the amount of the dose andthe frequency of administration, such as, for example, the timing of thenext dose. The term “dosage level” refers to the amount of the modifiedcells administered in relation to the body weight of the subject.

In certain embodiments the cells are in an animal, such as human,non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent.The subject may be, for example, an animal, such as a mammal, forexample, a human, non-human primate, cow, horse, pig, sheep, goat, dog,cat, or rodent. The subject may be, for example, human, for example, apatient suffering from an infectious disease, and/or a subject that isimmunocompromised, or is suffering from a hyperproliferative disease.

Thus, for example, in certain embodiments, the methods comprisedetermining the presence or absence of a tumor size increase and/orincrease in the number of tumor cells in a subject relative to the tumorsize and/or the number of tumor cells following administration of themodified cells or nucleic acid, and administering an additional dose ofthe modified cells or nucleic acid to the subject in the event thepresence of a tumor size increase and/or increase in the number of tumorcells is determined. The methods also comprise, for example, determiningthe presence or absence of an increase in CD19-expressing B cells in thesubject relative to the level of CD19-expressing B cells followingadministration of the modified cells or nucleic acid, and administeringan additional dose of the modified cells or nucleic acid to the subjectin the event the presence of an increase in CD19-expressing B cells inthe subject is determined. In these embodiments, for example, thepatient is initially treated with the therapeutic cells or nucleic acidaccording to the methods provided herein. Following the initialtreatment, the size of the tumor, the number of tumor cells, or thenumber of CD19-expressing B cells, for example, may decrease relative tothe time prior to the initial treatment. At a certain time after thisinitial treatment, the patient is again tested, or the patient may becontinually monitored for disease symptoms. If it is determined that thesize of the tumor, the number of tumor cells, or the number ofCD19-expressing B cells, for example, is increased relative to the timejust after the initial treatment, then the modified cells or nucleicacid may be administered for an additional dose. This monitoring andtreatment schedule may continue while noting that the therapeutic cellsthat express chimeric antigen receptors or chimeric stimulatingmolecules remain in the patient.

In other embodiments, following administration of the modified cells ornucleic acid, wherein the modified cells or nucleic acid express theinducible Caspase-9 polypeptide, in the event of a need to reduce thenumber of modified cells or in vivo modified cells, the multimericligand may be administered to the patient. In these embodiments, themethods comprise determining the presence or absence of a negativesymptom or condition, such as Graft vs Host Disease, or off targettoxicity, and administering a dose of the multimeric ligand. The methodsmay further comprise monitoring the symptom or condition andadministering an additional dose of the multimeric ligand in the eventthe symptom or condition persists. This monitoring and treatmentschedule may continue while the therapeutic cells that express chimericantigen receptors or chimeric stimulating molecules remain in thepatient.

An indication of adjusting or maintaining a subsequent drug dose, suchas, for example, a subsequent dose of the modified cells or nucleicacid, and/or the subsequent drug dosage, can be provided in anyconvenient manner. An indication may be provided in tabular form (e.g.,in a physical or electronic medium) in some embodiments. For example,the size of the tumor cell, or the number or level of tumor cells in asample may be provided in a table, and a clinician may compare thesymptoms with a list or table of stages of the disease. The clinicianthen can identify from the table an indication for subsequent drug dose.In certain embodiments, an indication can be presented (e.g., displayed)by a computer, after the symptoms are provided to the computer (e.g.,entered into memory on the computer). For example, this information canbe provided to a computer (e.g., entered into computer memory by a useror transmitted to a computer via a remote device in a computer network),and software in the computer can generate an indication for adjusting ormaintaining a subsequent drug dose, and/or provide the subsequent drugdose amount.

Once a subsequent dose is determined based on the indication, aclinician may administer the subsequent dose or provide instructions toadjust the dose to another person or entity. The term “clinician” asused herein refers to a decision maker, and a clinician is a medicalprofessional in certain embodiments. A decision maker can be a computeror a displayed computer program output in some embodiments, and a healthservice provider may act on the indication or subsequent drug dosedisplayed by the computer. A decision maker may administer thesubsequent dose directly (e.g., infuse the subsequent dose into thesubject) or remotely (e.g., pump parameters may be changed remotely by adecision maker).

Methods as presented herein include without limitation the delivery ofan effective amount of an activated cell, a nucleic acid, or anexpression construct encoding the same. An “effective amount” of theactivated cell, nucleic acid, or expression construct, generally, isdefined as that amount sufficient to detectably and repeatedly toachieve the stated desired result, for example, to ameliorate, reduce,minimize or limit the extent of the disease or its symptoms. Other morerigorous definitions may apply, including elimination, eradication orcure of disease. In some embodiments there may be a step of monitoringthe biomarkers, or other disease symptoms such as tumor size or tumorantigen expression, to evaluate the effectiveness of treatment and tocontrol toxicity.

In further embodiments, the expression construct and/or expressionvector can be utilized as a composition or substance that activatescells. Such a composition that “activates cells” or “enhances theactivity of cells” refers to the ability to stimulate one or moreactivities associated with cells. For example, a composition, such asthe expression construct or vector of the present methods, can stimulateupregulation of co-stimulating molecules on cells, induce nucleartranslocation of NF-κB in cells, activate toll-like receptors in cells,or other activities involving cytokines or chemokines.

The expression construct, expression vector and/or transduced cells canenhance or contribute to the effectiveness of a vaccine by, for example,enhancing the immunogenicity of weaker antigens such as highly purifiedor recombinant antigens, reducing the amount of antigen required for animmune response, reducing the frequency of immunization required toprovide protective immunity, improving the efficacy of vaccines insubjects with reduced or weakened immune responses, such as newborns,the aged, and immunocompromised individuals, and enhancing the immunityat a target tissue, such as mucosal immunity, or promote cell-mediatedor humoral immunity by eliciting a particular cytokine profile.

In certain embodiments, the cell is also contacted with an antigen.Often, the cell is contacted with the antigen ex vivo. Sometimes, thecell is contacted with the antigen in vivo. In some embodiments, thecell is in a subject and an immune response is generated against theantigen. Sometimes, the immune response is a cytotoxic T-lymphocyte(CTL) immune response. Sometimes, the immune response is generatedagainst a tumor antigen. In certain embodiments, the cell is activatedwithout the addition of an adjuvant.

In certain embodiments, the cell can be transduced ex vivo or in vivowith a nucleic acid that encodes the chimeric protein. The cell may besensitized to the antigen at the same time the cell is contacted withthe multimeric ligand, or the cell can be pre-sensitized to the antigenbefore the cell is contacted with the multimerization ligand. In someembodiments, the cell is contacted with the antigen ex vivo.

In some embodiments, the cell is transduced with the nucleic acid exvivo and administered to the subject by intradermal administration. Insome embodiments, the cell is transduced with the nucleic acid ex vivoand administered to the subject by subcutaneous administration.Sometimes, the cell is transduced with the nucleic acid ex vivo.Sometimes, the cell is transduced with the nucleic acid in vivo.

In certain embodiments, the cell is transduced with the nucleic acid exvivo and administered to the subject by intradermal administration, andsometimes the cell is transduced with the nucleic acid ex vivo andadministered to the subject by subcutaneous administration. The antigenmay be a tumor antigen, and the CTL immune response can be induced bymigration of the cell to a draining lymph node. A tumor antigen is anyantigen such as, for example, a peptide or polypeptide, that triggers animmune response in a host. The tumor antigen may be a tumor-associatedantigen, which is associated with a neoplastic tumor cell.

In some embodiments, an immunocompromised individual or subject is asubject that has a reduced or weakened immune response. Such individualsmay also include a subject that has undergone chemotherapy or any othertherapy resulting in a weakened immune system, a transplant recipient, asubject currently taking immunosuppressants, an aging individual, or anyindividual that has a reduced and/or impaired CD4 T helper cells. It iscontemplated that the present methods can be utilized to enhance theamount and/or activity of CD4 T helper cells in an immunocompromisedsubject.

Antigens

Chimeric antigen receptors bind to target antigens. When assaying T cellactivation in vitro or ex vivo, target antigens may be obtained orisolated from various sources. The target antigen, as used herein, is anantigen or immunological epitope on the antigen, which is crucial inimmune recognition and ultimate elimination or control of thedisease-causing agent or disease state in a mammal. The immunerecognition may be cellular and/or humoral. In the case of intracellularpathogens and cancer, immune recognition may, for example, be a Tlymphocyte response.

The target antigen may be derived or isolated from, for example, apathogenic microorganism such as viruses including HIV, (Korber et al,eds HIV Molecular Immunology Database, Los Alamos National Laboratory,Los Alamos, N. Mex. 1977) influenza, Herpes simplex, human papillomavirus (U.S. Pat. No. 5,719,054), Hepatitis B (U.S. Pat. No. 5,780,036),Hepatitis C (U.S. Pat. No. 5,709,995), EBV, Cytomegalovirus (CMV) andthe like. Target antigen may be derived or isolated from pathogenicbacteria such as, for example, from Chlamydia (U.S. Pat. No. 5,869,608),Mycobacteria, Legionella, Meningiococcus, Group A Streptococcus,Salmonella, Listeria, Hemophilus influenzae (U.S. Pat. No. 5,955,596)and the like).

Target antigen may be derived or isolated from, for example, pathogenicyeast including Aspergillus, invasive Candida (U.S. Pat. No. 5,645,992),Nocardia, Histoplasmosis, Cryptosporidia and the like.

Target antigen may be derived or isolated from, for example, apathogenic protozoan and pathogenic parasites including but not limitedto Pneumocystis carinii, Trypanosoma, Leishmania (U.S. Pat. No.5,965,242), Plasmodium (U.S. Pat. No. 5,589,343) and Toxoplasma gondii.

Target antigen includes an antigen associated with a preneoplastic orhyperplastic state. Target antigen may also be associated with, orcausative of cancer. Such target antigen may be, for example, tumorspecific antigen, tumor associated antigen (TAA) or tissue specificantigen, epitope thereof, and epitope agonist thereof. Such targetantigens include but are not limited to carcinoembryonic antigen (CEA)and epitopes thereof such as CAP-1, CAP-1-6D and the like (GenBankAccession No. M29540), MART-1 (Kawakarni et al, J. Exp. Med.180:347-352, 1994), MAGE-1 (U.S. Pat. No. 5,750,395), MAGE-3, GAGE (U.S.Pat. No. 5,648,226), GP-100 (Kawakami et al Proc. Nat'l Acad. Sci. USA91:6458-6462, 1992), MUC-1, MUC-2, point mutated ras oncogene, normaland point mutated p53 oncogenes (Hollstein et al Nucleic Acids Res.22:3551-3555, 1994), PSMA (Israeli et al Cancer Res. 53:227-230, 1993),tyrosinase (Kwon et al PNAS 84:7473-7477, 1987) TRP-1 (gp75) (Cohen etal Nucleic Acid Res. 18:2807-2808, 1990; U.S. Pat. No. 5,840,839),NY-ESO-1 (Chen et al PNAS 94: 1914-1918, 1997), TRP-2 (Jackson et alEMBOJ, 11:527-535, 1992), TAG72, KSA, CA-125, PSA, HER-2/neu/c-erb/B2,(U.S. Pat. No. 5,550,214), BRC-I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1,modifications of TAAs and tissue specific antigen, splice variants ofTAAs, epitope agonists, and the like. Other TAAs may be identified,isolated and cloned by methods known in the art such as those disclosedin U.S. Pat. No. 4,514,506. Target antigen may also include one or moregrowth factors and splice variants of each.

An antigen may be expressed more frequently in cancer cells than innon-cancer cells. The antigen may result from contacting the modifiedcell with a prostate specific membrane antigen, for example, a prostatespecific membrane antigen (PSMA) or fragment thereof.

Prostate antigen (PA001) is a recombinant protein consisting of theextracellular portion of PSMA antigen. PSMA is a ˜100 kDa (84 kDa beforeglycosylation, ˜180 kDa as dimer) type II membrane protein withneuropeptidase and folate hydrolase activities, but the true function ofPSMA is currently unclear. Carter R E, et al., Proc Natl Acad Sci USA.93: 749-53, 1996; Israeli R S, et al., Cancer Res. 53: 227-30, 1993;Pinto J T, et al., Clin Cancer Res. 2: 1445-51, 1996. Expression islargely, but not exclusively, prostate-specific and is maintained inadvanced and hormone refractory disease. Israeli R S, et al., CancerRes. 54: 1807-11, 1994. Weak non-prostatic detection in normal tissueshas also been seen in the salivary gland, brain, small intestines,duodenal mucosa, proximal renal tubules and neuroendocrine cells incolonic crypts. Silver D A, et al., Clin Cancer Res. 3: 81-5, 1997;Troyer J K, et al., Int J Cancer. 62: 552-8, 1995. Moreover, PSMA isup-regulated following androgen deprivation therapy (ADT). Wright G L,Jr., et al., Urology. 48: 326-34, 1996. While most PSMA is expressed asa cytoplasmic protein, the alternatively spliced transmembrane form isthe predominate form on the apical surface of neoplastic prostate cells.Su S L, et al., Cancer Res. 55: 1441-3, 1995; Israeli R S, et al.,Cancer Res. 54: 6306-10, 1994.

Moreover, PSMA is internalized following cross-linking and has been usedto internalize bound antibody or ligand complexed with radionucleotidesor viruses and other complex macromolecules. Liu H, et al., Cancer Res.58: 4055-60, 1998; Freeman L M, et al., Q J Nucl Med. 46: 131-7, 2002;Kraaij R, et al., Prostate. 62: 253-9, 2005. Bander and colleaguesdemonstrated that pretreatment of tumors with microtubule inhibitorsincreases aberrant basal surface targeting and antibody-mediatedinternalization of PSMA. Christiansen J J, et al., Mol Cancer Ther. 4:704-14, 2005. Tumor targeting may be facilitated by the observation ofectopic expression of PSMA in tumor vascular endothelium of not onlyprostate, but also renal and other tumors. Liu H, et al., Cancer Res.57: 3629-34, 1997; Chang S S, et al., Urology. 57: 801-5, 2001; Chang SS, et al., Clin Cancer Res. 5: 2674-81, 1999.

PSMA is not found in the vascular endothelial cells of correspondingbenign tissue. De la Taille A, et al., Cancer Detect Prev. 24: 579-88,2000. Although one early histological study of metastatic prostatedisease suggested that only ˜50% (8 of 18) of bone metastases (with 7 of8 lymph node metastases) expressed PSMA, the more sensitive reagent,177Lu-radiolabeled MoAb J591, targeted to the ectodomain of PSMA, couldtarget all known sites of bone and soft tissue metastasis in 30 of 30patients, suggesting near universal expression in advanced prostatedisease. Bander N. H., et al., J Clin Oncol. 23: 4591-601, 2005.

A prostate specific antigen, or PSA, is meant to include any antigenthat can induce an immune response, such as, for example, a cytotoxic Tlymphocyte response, against a PSA, for example, a PSMA, and may bespecifically recognized by any anti-PSA antibody. PSAs used in thepresent method are capable of being used to load the cell, as assayedusing conventional methods. Thus, “prostate specific antigen” or “PSA”may, for example, refer to a protein having the wild type amino acidsequence of a PSA, or a polypeptide that includes a portion of the a PSAprotein,

A prostate specific membrane antigen, or PSMA, is meant to include anyantigen that can induce an immune response, such as, for example, acytotoxic T lymphocyte response, against PSMA, and may be specificallyrecognized by an anti-PSMA antibody. PSMAs used in the present methodare capable of being used to load the cell, as assayed usingconventional methods. Thus, “prostate specific membrane antigen” or“PSMA” may, for example, refer to a protein having the wild type aminoacid sequence of PSMA, or a polypeptide that includes a portion of thePSMA protein, such as one encoded by SEQ ID NO: 3, or a portion of thenucleotide sequence of SEQ ID NO:3, or having the polypeptide of SEQ IDNO: 4, or a portion thereof. The term may also refer to, for example, apeptide having an amino acid sequence of a portion of SEQ ID NO: 4, orany peptide that may induce an immune response against PSMA. Alsoincluded are variants of any of the foregoing, including, for example,those having substitutions and deletions. Proteins, polypeptides, andpeptides having differential post-translational processing, such asdifferences in glycosylation, from the wild type PSMA, may also be usedin the present methods. Further, various sugar molecules that arecapable of inducing an immune response against PSMA, are alsocontemplated.

A PSA, for example, a PSMA, polypeptide may be used to load the modifiedcell. In certain embodiments, the modified cell is contacted with a PSMApolypeptide fragment having the amino acid sequence of SEQ ID NO: 4(e.g., encoded by the nucleotide sequence of SEQ ID NO: 3), or afragment thereof. In some embodiments, the PSA, for example, PSMApolypeptide fragment does not include the signal peptide sequence. Inother embodiments, the modified cell is contacted with a PSA, forexample, PSMA polypeptide fragment comprising substitutions or deletionsof amino acids in the polypeptide, and the fragment is sufficient toload cells.

A prostate specific protein antigen, or s PSPA, also referred to in thisspecification as a prostate specific antigen, or a PSA, is meant toinclude any antigen that can induce an immune response, such as, forexample, a cytotoxic T lymphocyte response, against a prostate specificprotein antigen. This includes, for example, a prostate specific proteinantigen or Prostate Specific Antigen. PSPAs used in the present methodare capable of being used to load the cell, as assayed usingconventional methods. Prostate Specific Antigen, or PSA, may, forexample, refer to a protein having the wild type amino acid sequence ofa PSA, or a polypeptide that includes a portion of the PSA protein,

A prostate specific membrane antigen, or PSMA, is meant to include anyantigen that can induce an immune response, such as, for example, acytotoxic T lymphocyte response, against PSMA, and may be specificallyrecognized by an anti-PSMA antibody. PSMAs used in the present methodare capable of being used to load the cell, as assayed usingconventional methods. Thus, “prostate specific membrane antigen” or“PSMA” may, for example, refer to a protein having the wild type aminoacid sequence of PSMA, or a polypeptide that includes a portion of thePSMA protein, such as one encoded by SEQ ID NO: 3, or a portion of thenucleotide sequence of SEQ ID NO:3, or having the polypeptide of SEQ IDNO: 4, or a portion thereof. The term may also refer to, for example, apeptide having an amino acid sequence of a portion of SEQ ID NO: 4, orany peptide that may induce an immune response against PSMA. Alsoincluded are variants of any of the foregoing, including, for example,those having substitutions and deletions. Proteins, polypeptides, andpeptides having differential post-translational processing, such asdifferences in glycosylation, from the wild type PSMA, may also be usedin the present methods. Further, various sugar molecules that arecapable of inducing an immune response against PSMA, are alsocontemplated.

A PSPA, for example, a PSMA, polypeptide may be used to load themodified cell. In certain embodiments, the modified cell is contactedwith a PSMA polypeptide fragment having the amino acid sequence of SEQID NO: 4 (e.g., encoded by the nucleotide sequence of SEQ ID NO: 3), ora fragment thereof. In some embodiments, the PSA, for example, PSMApolypeptide fragment does not include the signal peptide sequence. Inother embodiments, the modified cell is contacted with a PSPA, forexample, PSMA polypeptide fragment comprising substitutions or deletionsof amino acids in the polypeptide, and the fragment is sufficient toload cells.

A tumor antigen is any antigen such as, for example, a peptide orpolypeptide, that triggers an immune response in a host against a tumor.The tumor antigen may be a tumor-associated antigen, which is associatedwith a neoplastic tumor cell.

A prostate cancer antigen, or PCA, is any antigen such as, for example,a peptide or polypeptide, that triggers an immune response in a hostagainst a prostate cancer tumor. A prostate cancer antigen may, or maynot, be specific to prostate cancer tumors. A prostate cancer antigenmay also trigger immune responses against other types of tumors orneoplastic cells. A prostate cancer antigen includes, for example,prostate specific protein antigens, prostate specific antigens, andprostate specific membrane antigens.

The cell may be contacted with tumor antigen, such as PSA, for example,PSMA polypeptide, by various methods, including, for example, pulsingimmature DCs with unfractionated tumor lysates, MHC-eluted peptides,tumor-derived heat shock proteins (HSPs), tumor associated antigens(TAAs (peptides or proteins)), or transfecting DCs with bulk tumor mRNA,or mRNA coding for TAAs (reviewed in Gilboa, E. & Vieweg, J., ImmunolRev 199, 251-63 (2004); Gilboa, E, Nat Rev Cancer 4, 401-11 (2004)).

For organisms that contain a DNA genome, a gene encoding a targetantigen or immunological epitope thereof of interest is isolated fromthe genomic DNA. For organisms with RNA genomes, the desired gene may beisolated from cDNA copies of the genome. If restriction maps of thegenome are available, the DNA fragment that contains the gene ofinterest is cleaved by restriction endonuclease digestion by routinemethods. In instances where the desired gene has been previously cloned,the genes may be readily obtained from the available clones.Alternatively, if the DNA sequence of the gene is known, the gene can besynthesized by any of the conventional techniques for synthesis ofdeoxyribonucleic acids.

Genes encoding an antigen of interest can be amplified, for example, bycloning the gene into a bacterial host. For this purpose, variousprokaryotic cloning vectors can be used. Examples are plasmids pBR322,pUC and pEMBL.

The genes encoding at least one target antigen or immunological epitopethereof can be prepared for insertion into the plasmid vectors designedfor recombination with a virus by standard techniques. In general, thecloned genes can be excised from the prokaryotic cloning vector byrestriction enzyme digestion. In most cases, the excised fragment willcontain the entire coding region of the gene. The DNA fragment carryingthe cloned gene can be modified as needed, for example, to make the endsof the fragment compatible with the insertion sites of the DNA vectorsused for recombination with a virus, then purified prior to insertioninto the vectors at restriction endonuclease cleavage sites (cloningsites).

Antigen loading of cells, such as, for example, dendritic cells, withantigens may be achieved, for example, by contacting cells, such as, forexample, dendritic cells or progenitor cells with an antigen, forexample, by incubating the cells with the antigen. Loading may also beachieved, for example, by incubating DNA (naked or within a plasmidvector) or RNA that code for the antigen; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the antigen may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide. Antigens from cells or MHC molecules may be obtained byacid-elution or other methods (see Zitvogel L, et al., J Exp Med 1996.183:87-97). The cells may be transduced or transfected with the chimericprotein-encoding nucleotide sequence according to the present methodsbefore, after, or at the same time as the cells are loaded with antigen.In particular embodiments, antigen loading is subsequent to transductionor transfection.

In further embodiments, the transduced cell is transfected with tumorcell mRNA. The transduced transfected cell is administered to an animalto effect cytotoxic T lymphocytes and natural killer cell anti-tumorantigen immune response and regulated using dimeric FK506 and dimericFK506 analogs. The tumor cell mRNA may be, for example, mRNA from aprostate tumor cell.

In some embodiments, the transduced cell may be loaded by pulsing withtumor cell lysates. The pulsed transduced cells are administered to ananimal to effect cytotoxic T lymphocytes and natural killer cellanti-tumor antigen immune response and regulated using dimeric FK506 anddimeric FK506 analogs. The tumor cell lysate may be, for example, aprostate tumor cell lysate.

Immune Cells and Cytotoxic T Lymphocyte Response

T-lymphocytes may be activated by contact with the cell that comprisesthe expression vector discussed herein, where the cell has beenchallenged, transfected, pulsed, or electrofused with an antigen.

T cells express a unique antigen binding receptor on their membrane(T-cell receptor), which can only recognize antigen in association withmajor histocompatibility complex (MHC) molecules on the surface of othercells. There are several populations of T cells, such as T helper cellsand T cytotoxic cells. T helper cells and T cytotoxic cells areprimarily distinguished by their display of the membrane boundglycoproteins CD4 and CD8, respectively. T helper cells secret variouslymphokines, which are crucial for the activation of B cells, Tcytotoxic cells, macrophages and other cells of the immune system. Incontrast, a naïve CD8 T cell that recognizes an antigen-MHC complexproliferates and differentiates into an effector cell called a cytotoxicCD8 T lymphocyte (CTL). CTLs eliminate cells of the body displayingantigen, such as virus-infected cells and tumor cells, by producingsubstances that result in cell lysis.

CTL activity can be assessed by methods discussed herein, for example.For example, CTLs may be assessed in freshly isolated peripheral bloodmononuclear cells (PBMC), in a phytohaemaglutinin-stimulated IL-2expanded cell line established from PBMC (Bernard et al., AIDS,12(16):2125-2139, 1998) or by T cells isolated from a previouslyimmunized subject and restimulated for 6 days with DC infected with anadenovirus vector containing antigen using standard 4 hour 51Cr releasemicrotoxicity assays. One type of assay uses cloned T-cells. ClonedT-cells have been tested for their ability to mediate both perforin andFas ligand-dependent killing in redirected cytotoxicity assays (Simpsonet al., Gastroenterology, 115(4):849-855, 1998). The cloned cytotoxic Tlymphocytes displayed both Fas- and perforin-dependent killing.Recently, an in vitro dehydrogenase release assay has been developedthat takes advantage of a new fluorescent amplification system (Page,B., et al., Anticancer Res. 1998 July-August; 18(4A):2313-6). Thisapproach is sensitive, rapid, and reproducible and may be usedadvantageously for mixed lymphocyte reaction (MLR). It may easily befurther automated for large-scale cytotoxicity testing using cellmembrane integrity, and is thus considered. In another fluorometricassay developed for detecting cell-mediated cytotoxicity, thefluorophore used is the non-toxic molecule AlamarBlue (Nociari et al.,J. Immunol. Methods, 213(2): 157-167, 1998). The AlamarBlue isfluorescently quenched (i.e., low quantum yield) until mitochondrialreduction occurs, which then results in a dramatic increase in theAlamarBlue fluorescence intensity (i.e., increase in the quantum yield).This assay is reported to be extremely sensitive, specific and requiresa significantly lower number of effector cells than the standard ⁵¹Crrelease assay.

Other immune cells that can be induced by the present methods includenatural killer cells (NK). NKs are lymphoid cells that lackantigen-specific receptors and are part of the innate immune system.Typically, infected cells are usually destroyed by T cells alerted byforeign particles bound to the cell surface MHC. However, virus-infectedcells signal infection by expressing viral proteins that are recognizedby antibodies. These cells can be killed by NKs. In tumor cells, if thetumor cells lose expression of MHC I molecules, then it may besusceptible to NKs.

Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—expression constructs, expressionvectors, fused proteins, transduced cells, activated T cells, transducedand loaded T cells—in a form appropriate for the intended application.Generally, this will entail preparing compositions that are essentiallyfree of pyrogens, as well as other impurities that could be harmful tohumans or animals.

The multimeric ligand, such as, for example, AP1903, may be delivered,for example at doses of about 0.01 to 1 mg/kg subject weight, of about0.05 to 0.5 mg/kg subject weight, 0.1 to 2 mg/kg subject weight, ofabout 0.05 to 1.0 mg/kg subject weight, of about 0.1 to 5 mg/kg subjectweight, of about 0.2 to 4 mg/kg subject weight, of about 0.3 to 3 mg/kgsubject weight, of about 0.3 to 2 mg/kg subject weight, or about 0.3 to1 mg/kg subject weight, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10mg/kg subject weight. In some embodiments, the ligand is provided at 0.4mg/kg per dose, for example at a concentration of 5 mg/mL. Vials orother containers may be provided containing the ligand at, for example,a volume per vial of about 0.25 ml to about 10 ml, for example, about0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,8.5, 9, 9.5, or 10 ml, for example, about 2 ml.

AP1903 for Injection

AP1903 API is manufactured by Alphora Research Inc. and AP1903 DrugProduct for Injection is made by Formatech Inc. It is formulated as a 5mg/mL solution of AP1903 in a 25% solution of the non-ionic solubilizerSolutol H S 15 (250 mg/mL, BASF). At room temperature, this formulationis a clear, slightly yellow solution. Upon refrigeration, thisformulation undergoes a reversible phase transition, resulting in amilky solution. This phase transition is reversed upon re-warming toroom temperature. The fill is 2.33 mL in a 3 mL glass vial (˜10 mgAP1903 for Injection total per vial).

AP1903 is removed from the refrigerator the night before the patient isdosed and stored at a temperature of approximately 21° C. overnight, sothat the solution is clear prior to dilution. The solution is preparedwithin 30 minutes of the start of the infusion in glass or polyethylenebottles or non-DEHP bags and stored at approximately 21° C. prior todosing.

All study medication is maintained at a temperature between 2 degrees C.and 8 degrees C., protected from excessive light and heat, and stored ina locked area with restricted access.

Administration

In one example, patients are administered a single fixed dose of AP1903for Injection (0.4 mg/kg) via IV infusion over 2 hours, using anon-DEHP, non-ethylene oxide sterilized infusion set. The dose of AP1903is calculated individually for all patients, and is not be recalculatedunless body weight fluctuates by 10%. The calculated dose is diluted in100 mL in 0.9% normal saline before infusion.

Patients are observed for 15 minutes following the end of the infusionfor untoward adverse effects. One may generally desire to employappropriate salts and buffers to render delivery vectors stable andallow for uptake by target cells. Buffers also may be employed whenrecombinant cells are introduced into a patient. Aqueous compositionscomprise an effective amount of the vector to cells, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions also are referred to as inocula. The phrase“pharmaceutically or pharmacologically acceptable” refers to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Apharmaceutically acceptable carrier includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is known. Exceptinsofar as any conventional media or agent is incompatible with thevectors or cells, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The active compositions may include classic pharmaceutical preparations.Administration of these compositions will be via any common route solong as the target tissue is available via that route. This includes,for example, oral, nasal, buccal, rectal, vaginal or topical.Alternatively, administration may be by orthotopic, intradermal,subcutaneous, intramuscular, intraperitoneal or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions, discussed herein.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form is sterile and is fluid to the extentthat easy syringability exists. It is stable under the conditions ofmanufacture and storage and is preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In certainexamples, isotonic agents, for example, sugars or sodium chloride may beincluded. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

For oral administration, the compositions may be incorporated withexcipients and used in the form of non-ingestible mouthwashes anddentifrices. A mouthwash may be prepared incorporating the activeingredient in the required amount in an appropriate solvent, such as asodium borate solution (Dobell's Solution). Alternatively, the activeingredient may be incorporated into an antiseptic wash containing sodiumborate, glycerin and potassium bicarbonate. The active ingredient alsomay be dispersed in dentifrices, including, for example: gels, pastes,powders and slurries. The active ingredient may be added in atherapeutically effective amount to a paste dentifrice that may include,for example, water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions may be formulated in a neutral or salt form.Pharmaceutically acceptable salts include, for example, the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution may be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media can be employed. For example, onedosage could be dissolved in 1 ml of isotonic NaCl solution and eitheradded to 1000 ml of hypodermoclysis fluid or injected at the proposedsite of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations may meet sterility,pyrogenicity, and general safety and purity standards as required by FDAOffice of Biologics standards.

The administration schedule may be determined as appropriate for thepatient and may, for example, comprise a dosing schedule where thenucleic acid is administered at week 0, followed by induction byadministration of the chemical inducer of dimerization, followed byadministration of additional inducer when needed to obtain an effectivetherapeutic result or, for example, at 2, 4, 6, 8, 10, 12, 14, 16, 18,20 intervals thereafter for a total of, for example, 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, or 30, 40, 50, 60, 70, 80, 90, or 100weeks.

The administration schedule may be determined as appropriate for thepatient and may, for example, comprise a dosing schedule where thenucleic acid-transduced T cell or other cell is administered at week 0,followed by induction by administration of the chemical inducer ofdimerization, followed by administration of additional inducer whenneeded to obtain an effective therapeutic result or, for example, at 2,4, 6, 8, 10, 12, 14, 16, 18, 20 intervals thereafter for a total of, forexample, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30, 40,50, 60, 70, 80, 90, or 100 weeks.

Although for administration of transduced T cells, one dose is likely tobe sufficient, T cells may be provided more than once, or other cells,such as the non-dendritic cells and non-B cells discussed herein mayalso be administered multiple times. In addition, nucleic acids targetedto the non-T cell aspects of the present technology may also beadministered more than one time for optimum therapeutic efficacy.Therefore, for example, the administration schedule may be determined asappropriate for the patient and may, for example, comprise a dosingschedule where the nucleic acid or nucleic acid-transduced cell isadministered at week 0, followed by administration of additional nucleicacid or nucleic acid-transduced cell and inducer at 2 week intervalsthereafter for a total of, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, or 30 weeks.

Administration of a dose of cells may occur in one session, or in morethan one session. If needed, the method may further include additionalleukaphereses to obtain more cells to be used in treatment.

Methods for Treating a Disease

The present methods also encompass methods of treatment or prevention ofa disease caused by pathogenic microorganisms and/or ahyperproliferative disease.

Diseases that may be treated or prevented include diseases caused byviruses, bacteria, yeast, parasites, protozoa, cancer cells and thelike. The pharmaceutical composition (transduced T cells, expressionvector, expression construct, etc.) may be used as a generalized immuneenhancer (T cell activating composition or system) and as such hasutility in treating diseases. Exemplary diseases that can be treatedand/or prevented include, but are not limited, to infections of viraletiology such as HIV, influenza, Herpes, viral hepatitis, Epstein Bar,polio, viral encephalitis, measles, chicken pox, Papilloma virus etc.;or infections of bacterial etiology such as pneumonia, tuberculosis,syphilis, etc.; or infections of parasitic etiology such as malaria,trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc.

Preneoplastic or hyperplastic states which may be treated or preventedusing the pharmaceutical composition (transduced T cells, expressionvector, expression construct, etc.) include but are not limited topreneoplastic or hyperplastic states such as colon polyps, Crohn'sdisease, ulcerative colitis, breast lesions and the like.

Cancers, including solid tumors, which may be treated using thepharmaceutical composition include, but are not limited to primary ormetastatic melanoma, adenocarcinoma, squamous cell carcinoma,adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer,liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemias,uterine cancer, breast cancer, prostate cancer, ovarian cancer,pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC,bladder cancer, cervical cancer and the like.

Other hyperproliferative diseases, including solid tumors, that may betreated using the T cell and other therapeutic cell activation systempresented herein include, but are not limited to rheumatoid arthritis,inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasiaand prostatic intraepithelial neoplasia), carcinoma in situ, oral hairyleukoplakia, or psoriasis.

In the method of treatment, the administration of the pharmaceuticalcomposition (expression construct, expression vector, fused protein,transduced cells, and activated T cells, transduced and loaded T cells)may be for either “prophylactic” or “therapeutic” purpose. When providedprophylactically, the pharmaceutical composition is provided in advanceof any symptom. The prophylactic administration of pharmaceuticalcomposition serves to prevent or ameliorate any subsequent infection ordisease. When provided therapeutically, the pharmaceutical compositionis provided at or after the onset of a symptom of infection or disease.Thus the compositions presented herein may be provided either prior tothe anticipated exposure to a disease-causing agent or disease state orafter the initiation of the infection or disease. Thus provided hereinare methods for prophylactic treatment of solid tumors such as thosefound in cancer, or for example, but not limited to, prostate cancer,using the nucleic acids and cells discussed herein. For example, methodsare provided of prophylactically preventing or reducing the size of atumor in a subject comprising administering a the nucleic acids or cellsdiscussed herein, whereby the nucleic acids or cells are administered inan amount effect to prevent or reduce the size of a tumor in a subject.

Solid tumors from any tissue or organ may be treated using the presentmethods, including, for example, any tumor expressing PSA, for example,PSMA, in the vasculature, for example, solid tumors present in, forexample, lungs, bone, liver, prostate, or brain, and also, for example,in breast, ovary, bowel, testes, colon, pancreas, kidney, bladder,neuroendocrine system, soft tissue, boney mass, and lymphatic system.Other solid tumors that may be treated include, for example,glioblastoma, and malignant myeloma.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of pharmaceutical compositioncalculated to produce the desired immunogenic effect in association withthe required diluent. The specifications for the unit dose of aninoculum are dictated by and are dependent upon the uniquecharacteristics of the pharmaceutical composition and the particularimmunologic effect to be achieved.

An effective amount of the pharmaceutical composition would be theamount that achieves this selected result of enhancing the immuneresponse, and such an amount could be determined. For example, aneffective amount of for treating an immune system deficiency could bethat amount necessary to cause activation of the immune system,resulting in the development of an antigen specific immune response uponexposure to antigen. The term is also synonymous with “sufficientamount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One can empiricallydetermine the effective amount of a particular composition presentedherein without necessitating undue experimentation. Thus, for example,in one embodiment, the transduced T cells or other cells areadministered to a subject in an amount effective to, for example, inducean immune response, or, for example, to reduce the size of a tumor orreduce the amount of tumor vasculature.

In some embodiments, multiple doses of modified cells are administeredto the subject, with an escalation of dosage levels among the multipledoses. In some embodiments, the escalation of dosage levels increasesthe level of CAR-T cell activity, and therefore increases thetherapeutic effect, such as, for example, the reduction in the amount orconcentration of target cells, such as, for example, tumor cells.

In some embodiments, personalized treatment is provided wherein thestage or level of the disease or condition is determined beforeadministration of the modified cells, before the administration of anadditional dose of the modified cells, or in determining method anddosage involved in the administration of the modified cells. Thesemethods may be used in any of the methods of the present application.Where these methods of assessing the patient before administering themodified cells are discussed in the context of, for example, thetreatment of a subject with a solid tumor, it is understood that thesemethods may be similarly applied to the treatment of other conditionsand diseases. Thus, for example, in some embodiments of the presentapplication, the method comprises administering the modified cells ofthe present application to a subject, and further comprises determiningthe appropriate dose of modified cells to achieve the effective level ofreduction of tumor size. The amount of cells may be determined, forexample, based on the subject's clinical condition, weight, and/orgender or other relevant physical characteristic. By controlling theamount of modified cells administered to the subject, the likelihood ofadverse events such as, for example, a cytokine storm may be reduced.

The term “dosage” is meant to include both the amount of the dose andthe frequency of administration, such as, for example, the timing of thenext dose. For example, to induce the chimeric Caspase-9 polypeptide,the term “dosage level” refers to the amount of the multimeric ligandadministered in relation to the body weight of the subject. Thusincreasing the dosage level would mean increasing the amount of theligand administered relative to the subject's weight. In addition,increasing the concentration of the dose administered, such as, forexample, when the multimeric ligand is administered using a continuousinfusion pump would mean that the concentration administered (and thusthe amount administered) per minute, or second, is increased.

An indication of adjusting or maintaining a subsequent drug dose, suchas, for example, a subsequence dose of the multimeric ligand, and/or thesubsequent drug dosage, can be provided in any convenient manner. Anindication may be provided in tabular form (e.g., in a physical orelectronic medium) in some embodiments. For example, the disease orcondition symptoms may be provided in a table, and a clinician maycompare the symptoms with a list or table of stages of the disease. Theclinician then can identify from the table an indication for subsequentdrug dose. In certain embodiments, an indication can be presented (e.g.,displayed) by a computer, after the symptoms or the stage is provided tothe computer (e.g., entered into memory on the computer). For example,this information can be provided to a computer (e.g., entered intocomputer memory by a user or transmitted to a computer via a remotedevice in a computer network), and software in the computer can generatean indication for adjusting or maintaining a subsequent drug dose,and/or provide the subsequent drug dose amount.

Once a subsequent dose is determined based on the indication, aclinician may administer the subsequent dose or provide instructions toadjust the dose to another person or entity. The term “clinician” asused herein refers to a decision maker, and a clinician is a medicalprofessional in certain embodiments. A decision maker can be a computeror a displayed computer program output in some embodiments, and a healthservice provider may act on the indication or subsequent drug dosedisplayed by the computer. A decision maker may administer thesubsequent dose directly (e.g., infuse the subsequent dose into thesubject) or remotely (e.g., pump parameters may be changed remotely by adecision maker).

Methods as presented herein include without limitation the delivery ofan effective amount of an activated cell, a nucleic acid, or anexpression construct encoding the same. An “effective amount” of thepharmaceutical composition, generally, is defined as that amountsufficient to detectably and repeatedly to achieve the stated desiredresult, for example, to ameliorate, reduce, minimize or limit the extentof the disease or its symptoms. Other more rigorous definitions mayapply, including elimination, eradication or cure of disease. In someembodiments there may be a step of monitoring the biomarkers to evaluatethe effectiveness of treatment and to control toxicity.

A. Genetic Based Therapies

In certain embodiments, a cell is provided with an expression constructcapable of providing a co-stimulating polypeptide, such as thosediscussed herein, and, for example, in a T cell. The lengthy discussionof expression vectors and the genetic elements employed therein isincorporated into this section by reference. In certain examples, theexpression vectors may be viral vectors, such as adenovirus,adeno-associated virus, herpes virus, vaccinia virus and retrovirus. Inanother example, the vector may be a lysosomal-encapsulated expressionvector.

Gene delivery may be performed in both in vivo and ex vivo situations.For viral vectors, one generally will prepare a viral vector stock.Examples of viral vector-mediated gene delivery ex vivo and in vivo arepresented in the present application. For in vivo delivery, depending onthe kind of virus and the titer attainable, one will deliver, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, or 9×10⁴, 1, 2, 3, 4, 5, 6, 7, 8,or 9×10⁵, 1, 2, 3, 4, 5, 6, 7, 8, or 9×10⁶, 1, 2, 3, 4, 5, 6, 7, 8, or9×10⁷, 1, 2, 3, 4, 5, 6, 7, 8, or 9×10⁸, 1, 2, 3, 4, 5, 6, 7, 8, or9×10⁹, 1, 2, 3, 4, 5, 6, 7, 8, or 9×10¹⁰, 1, 2, 3, 4, 5, 6, 7, 8, or9×10¹¹ or 1, 2, 3, 4, 5, 6, 7, 8, or 9×10¹² infectious particles to thepatient. Similar figures may be extrapolated for liposomal or othernon-viral formulations by comparing relative uptake efficiencies.Formulation as a pharmaceutically acceptable composition is discussedbelow. The multimeric ligand, such as, for example, AP1903, may bedelivered, for example at doses of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10mg/kg subject weight.

B. Cell Based Therapy

Another therapy that is contemplated is the administration of transducedT cells. The T cells may be transduced in vitro. Formulation as apharmaceutically acceptable composition is discussed herein.

In cell based therapies, the transduced cells may be, for example,transfected with target antigen nucleic acids, such as mRNA or DNA orproteins; pulsed with cell lysates, proteins or nucleic acids; orelectrofused with cells. The cells, proteins, cell lysates, or nucleicacid may derive from cells, such as tumor cells or other pathogenicmicroorganism, for example, viruses, bacteria, protozoa, etc.

C. Combination Therapies

In order to increase the effectiveness of the expression vectorspresented herein, it may be desirable to combine these compositions andmethods with an agent effective in the treatment of the disease.

In certain embodiments, anti-cancer agents may be used in combinationwith the present methods. An “anti-cancer” agent is capable ofnegatively affecting cancer in a subject, for example, by killing one ormore cancer cells, inducing apoptosis in one or more cancer cells,reducing the growth rate of one or more cancer cells, reducing theincidence or number of metastases, reducing a tumor's size, inhibiting atumor's growth, reducing the blood supply to a tumor or one or morecancer cells, promoting an immune response against one or more cancercells or a tumor, preventing or inhibiting the progression of a cancer,or increasing the lifespan of a subject with a cancer. Anti-canceragents include, for example, chemotherapy agents (chemotherapy),radiotherapy agents (radiotherapy), a surgical procedure (surgery),immune therapy agents (immunotherapy), genetic therapy agents (genetherapy), hormonal therapy, other biological agents (biotherapy) and/oralternative therapies.

In further embodiments antibiotics can be used in combination with thepharmaceutical composition to treat and/or prevent an infectiousdisease. Such antibiotics include, but are not limited to, amikacin,aminoglycosides (e.g., gentamycin), amoxicillin, amphotericin B,ampicillin, antimonials, atovaquone sodium stibogluconate, azithromycin,capreomycin, cefotaxime, cefoxitin, ceftriaxone, chloramphenicol,clarithromycin, clindamycin, clofazimine, cycloserine, dapsone,doxycycline, ethambutol, ethionamide, fluconazole, fluoroquinolones,isoniazid, itraconazole, kanamycin, ketoconazole, minocycline,ofloxacin), para-aminosalicylic acid, pentamidine, polymixin definsins,prothionamide, pyrazinamide, pyrimethamine sulfadiazine, quinolones(e.g., ciprofloxacin), rifabutin, rifampin, sparfloxacin, streptomycin,sulfonamides, tetracyclines, thiacetazone,trimethaprim-sulfamethoxazole, viomycin or combinations thereof.

More generally, such an agent would be provided in a combined amountwith the expression vector effective to kill or inhibit proliferation ofa cancer cell and/or microorganism. This process may involve contactingthe cell(s) with an agent(s) and the pharmaceutical composition at thesame time or within a period of time wherein separate administration ofthe pharmaceutical composition and an agent to a cell, tissue ororganism produces a desired therapeutic benefit. This may be achieved bycontacting the cell, tissue or organism with a single composition orpharmacological formulation that includes both the pharmaceuticalcomposition and one or more agents, or by contacting the cell with twoor more distinct compositions or formulations, wherein one compositionincludes the pharmaceutical composition and the other includes one ormore agents.

The terms “contacted” and “exposed,” when applied to a cell, tissue ororganism, are used herein to describe the process by which thepharmaceutical composition and/or another agent, such as for example achemotherapeutic or radiotherapeutic agent, are delivered to a targetcell, tissue or organism or are placed in direct juxtaposition with thetarget cell, tissue or organism. To achieve cell killing or stasis, thepharmaceutical composition and/or additional agent(s) are delivered toone or more cells in a combined amount effective to kill the cell(s) orprevent them from dividing. The administration of the pharmaceuticalcomposition may precede, be concurrent with and/or follow the otheragent(s) by intervals ranging from minutes to weeks. In embodimentswhere the pharmaceutical composition and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the times ofeach delivery, such that the pharmaceutical composition and agent(s)would still be able to exert an advantageously combined effect on thecell, tissue or organism. For example, in such instances, it iscontemplated that one may contact the cell, tissue or organism with two,three, four or more modalities substantially simultaneously (i.e.,within less than about a minute) with the pharmaceutical composition. Inother aspects, one or more agents may be administered within of fromsubstantially simultaneously, about 1 minute, to about 24 hours to about7 days to about 1 to about 8 weeks or more, and any range derivabletherein, prior to and/or after administering the expression vector. Yetfurther, various combination regimens of the pharmaceutical compositionpresented herein and one or more agents may be employed.

In some embodiments, the chemotherapeutic agent may be a lymphodepletingchemotherapeutic. In other examples, the chemotherapeutic agent may beTaxotere (docetaxel), or another taxane, such as, for example,cabazitaxel. The chemotherapeutic may be administered before, during, orafter treatment with the cells and inducer. For example, thechemotherapeutic may be administered about 1 year, 11, 10, 9, 8, 7, 6,5, or 4 months, or 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,3, 2, weeks or 1 week prior to administering the first dose of activatednucleic acid. Or, for example, the chemotherapeutic may be administeredabout 1 week or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,or 18 weeks or 4, 5, 6, 7, 8, 9, 10, or 11 months or 1 year afteradministering the first dose of cells or inducer.

Administration of a chemotherapeutic agent may comprise theadministration of more than one chemotherapeutic agent. For example,cisplatin may be administered in addition to Taxotere or other taxane,such as, for example, cabazitaxel.

Generating an Immune Response Targeted to a Specific Tumor or Disease

Chimeric antigen receptors (CARs) are artificial receptors designed toconvey antigen specificity to T cells. They include an antigen-specificcomponent, a transmembrane component, and an intracellular componentselected to activate the T cell and provide specific immunity. They mayfurther comprise a stalk polypeptide, along with the transmembranecomponent. Chimeric antigen receptor-expressing T cells may be used invarious therapies, including cancer therapies.

The T cells and other cells transduced with the inducible CD40,inducible MyD88, or the inducible MyD88/CD40 may also be transduced witha nucleic acid coding for a chimeric antigen receptor, or CAR. Thechimeric antigen receptor may be selected to target tumor antigenspresent on the surface of the tumor to be treated, or other antigensassociated with disease. Activated T cells expressing the chimericantigen receptor would then target tumors, or other diseases. TransducedT cells may also include memory T cells, which would maintain the immunedefense against the particular tumor or disease. After administration ofthe modified T cells, modified memory T cells may be present in thesubject.

Optimized and Personalized Therapeutic Treatment

Treatment for solid tumor cancers, including, for example, prostatecancer, may be optimized by determining the concentration of IL-6,IL6-sR, or VCAM-1 during the course of treatment. IL-6 refers tointerleukin 6. IL-6sR refers to the IL-6 soluble receptor, the levels ofwhich often correlate closely with levels of IL-6. VCAM-1 refers tovascular cell adhesion molecule. Different patients having differentstages or types of cancer may react differently to various therapies.The response to treatment may be monitored by following the IL-6,IL-6sR, or VCAM-1 concentrations or levels in various body fluids ortissues. The determination of the concentration, level, or amount of apolypeptide, such as, IL-6, IL-6sR, or VCAM-1, may include detection ofthe full length polypeptide, or a fragment or variant thereof. Thefragment or variant may be sufficient to be detected by, for example,immunological methods, mass spectrometry, nucleic acid hybridization,and the like. Optimizing treatment for individual patients may help toavoid side effects as a result of overdosing, may help to determine whenthe treatment is ineffective and to change the course of treatment, ormay help to determine when doses may be increased. Technology discussedherein optimizes therapeutic methods for treating solid tumor cancers byallowing a clinician to track a biomarker, such as, for example, IL-6,IL-6sR, or VCAM-1, and determine whether a subsequent dose of a drug orvaccine for administration to a subject may be maintained, reduced orincreased, and to determine the timing for the subsequent dose.

Treatment for solid tumor cancers, including, for example, prostatecancer, may also be optimized by determining the concentration ofurokinase-type plasminogen activator receptor (uPAR), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), or vascular endothelialgrowth factor (VEGF) during the course of treatment. Different patientshaving different stages or types of cancer may react differently tovarious therapies. The levels of uPAR, HGF, EGF, and VEGF over thecourse of treatment for subject 1003 were measured. Subject 1003 showssystemic perturbation of hypoxic factors in serum, which may indicate apositive response to treatment. Without limiting the interpretation ofthis observation, this may indicate the secretion of hypoxic factors bytumors in response to treatment. Thus, the response to treatment may bemonitored, for example, by following the uPAR, HGF, EGF, or VEGFconcentrations or levels in various body fluids or tissues. Thedetermination of the concentration, level, or amount of a polypeptide,such as, uPAR, HGF, EGF, or VEGF may include detection of the fulllength polypeptide, or a fragment or variant thereof. The fragment orvariant may be sufficient to be detected by, for example, immunologicalmethods, mass spectrometry, nucleic acid hybridization, and the like.Optimizing treatment for individual patients may help to avoid sideeffects as a result of overdosing, may help to determine when thetreatment is ineffective and to change the course of treatment, or mayhelp to determine when doses may be increased. Technology discussedherein optimizes therapeutic methods for treating solid tumor cancers byallowing a clinician to track a biomarker, such as, for example, uPAR,HGF, EGF, or VEGF, and determine whether a subsequent dose of a drug orvaccine for administration to a subject may be maintained, reduced orincreased, and to determine the timing for the subsequent dose.

For example, it has been determined that amount or concentration ofcertain biomarkers changes during the course of treatment of solidtumors. Predetermined target levels of such biomarkers, or biomarkerthresholds may be identified in normal subject, are provided, whichallow a clinician to determine whether a subsequent dose of a drugadministered to a subject in need thereof, such as a subject with asolid tumor, such as, for example, a prostate tumor, may be increased,decreased or maintained. A clinician can make such a determination basedon whether the presence, absence or amount of a biomarker is below,above or about the same as a biomarker threshold, respectively, incertain embodiments.

For example, determining that an over-represented biomarker level issignificantly reduced and/or that an under-represented biomarker levelis significantly increased after drug treatment or vaccination providesan indication to a clinician that an administered drug is exerting atherapeutic effect. By “level” is meant the concentration of thebiomarker in a fluid or tissue, or the absolute amount in a tissue.Based on such a biomarker determination, a clinician could make adecision to maintain a subsequent dose of the drug or raise or lower thesubsequent dose, including modifying the timing of administration. Theterm “drug” includes traditional pharmaceuticals, such as smallmolecules, as well as biologics, such as nucleic acids, antibodies,proteins, polypeptides, modified cells and the like. In another example,determining that an over-represented biomarker level is notsignificantly reduced and/or that an under-represented biomarker levelis not significantly increased provides an indication to a clinicianthat an administered drug is not significantly exerting a therapeuticeffect. Based on such a biomarker determination, a clinician could makea decision to increase a subsequent dose of the drug. Given that drugscan be toxic to a subject and exert side effects, methods providedherein optimize therapeutic approaches as they provide the clinicianwith the ability to “dial in” an efficacious dosage of a drug andminimize side effects. In specific examples, methods provided hereinallow a clinician to “dial up” the dose of a drug to a therapeuticallyefficacious level, where the dialed up dosage is below a toxic thresholdlevel. Accordingly, treatment methods discussed herein enhance efficacyand reduce the likelihood of toxic side effects.

Cytokines are a large and diverse family of polypeptide regulatorsproduced widely throughout the body by cells of diverse origin.Cytokines are small secreted proteins, including peptides andglycoproteins, which mediate and regulate immunity, inflammation, andhematopoiesis. They are produced de novo in response to an immunestimulus. Cytokines generally (although not always) act over shortdistances and short time spans and at low concentration. They generallyact by binding to specific membrane receptors, which then signal thecell via second messengers, often tyrosine kinases, to alter cellbehavior (e.g., gene expression). Responses to cytokines include, forexample, increasing or decreasing expression of membrane proteins(including cytokine receptors), proliferation, and secretion of effectormolecules.

The term “cytokine” is a general description of a large family ofproteins and glycoproteins. Other names include lymphokine (cytokinesmade by lymphocytes), monokine (cytokines made by monocytes), chemokine(cytokines with chemotactic activities), and interleukin (cytokines madeby one leukocyte and acting on other leukocytes). Cytokines may act oncells that secrete them (autocrine action), on nearby cells (paracrineaction), or in some instances on distant cells (endocrine action).

Examples of cytokines include, without limitation, interleukins (e.g.,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and the like),interferons (e.g., IFN-α, IFN-β and the like), tumor necrosis factors(e.g., TNF-α, TNF-β and the like), lymphokines, monokines andchemokines; growth factors (e.g., transforming growth factors (e.g.,TGF-α, TGF-β and the like)); colony-stimulating factors (e.g. GM-CSF,granulocyte colony-simulating factor (G-CSF) etc.); and the like.

A cytokine often acts via a cell-surface receptor counterpart.Subsequent cascades of intracellular signaling then alter cellfunctions. This signaling may include upregulation and/or downregulationof several genes and their transcription factors, resulting in theproduction of other cytokines, an increase in the number of surfacereceptors for other molecules, or the suppression of their own effect byfeedback inhibition.

VCAM-1 (vascular cell adhesion molecule-1, also called CD106), containssix or seven immunoglobulin domains and is expressed on both large andsmall vessels only after the endothelial cells are stimulated bycytokines. Thus, VCAM-1 expression is a marker for cytokine expression.

Cytokines may be detected as full-length (e.g., whole) proteins,polypeptides, metabolites, messenger RNA (mRNA), complementary DNA(cDNA), and various intermediate products and fragments of the foregoing(e.g., cleavage products (e.g., peptides, mRNA fragments)). For example,IL-6 protein may be detected as the complete, full-length molecule or asany fragment large enough to provide varying levels of positiveidentification. Such a fragment may comprise amino acids numbering lessthan 10, from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 150,from 150 to 200 and above. Likewise, VCAM-1 protein can be detected asthe complete, full-length amino acid molecule or as any fragment largeenough to provide varying levels of positive identification. Such afragment may comprise amino acids numbering less than 10, from 10 to 20,from 20 to 50, from 50 to 100, from 100 to 150 and above.

In certain embodiments, cytokine mRNA may be detected by targeting acomplete sequence or any sufficient fragment for specific detection. AmRNA fragment may include fewer than 10 nucleotides or any largernumber. A fragment may comprise the 3′ end of the mRNA strand with anyportion of the strand, the 5′ end with any portion of the strand, andany center portion of the strand.

Detection may be performed using any suitable method, including, withoutlimitation, mass spectrometry (e.g., matrix-assisted laser desorptionionization mass spectrometry (MALDI-MS), electrospray mass spectrometry(ES-MS)), electrophoresis (e.g., capillary electrophoresis), highperformance liquid chromatography (HPLC), nucleic acid affinity (e.g.,hybridization), amplification and detection (e.g., real-time orreverse-transcriptase polymerase chain reaction (RT-PCR)), and antibodyassays (e.g., antibody array, enzyme-linked immunosorbant assay(ELISA)). Examples of IL-6 and other cytokine assays include, forexample, those provided by Millipore, Inc., (Milliplex HumanCytokine/Chemokine Panel). Examples of IL6-sR assays include, forexample, those provided by Invitrogen, Inc. (Soluble IL-6R: (InvitrogenLuminex® Bead-based assay)). Examples of VCAM-1 assays include, forexample, those provided by R & D Systems ((CD106) ELISA development Kit,DuoSet from R&D Systems (# DY809)).

Sources of Biomarkers

The presence, absence or amount of a biomarker can be determined withina subject (e.g., in situ) or outside a subject (e.g., ex vivo). In someembodiments, presence, absence or amount of a biomarker can bedetermined in cells (e.g., differentiated cells, stem cells), and incertain embodiments, presence, absence or amount of a biomarker can bedetermined in a substantially cell-free medium (e.g., in vitro). Theterm “identifying the presence, absence or amount of a biomarker in asubject” as used herein refers to any method known in the art forassessing the biomarker and inferring the presence, absence or amount inthe subject (e.g., in situ, ex vivo or in vitro methods).

A fluid or tissue sample often is obtained from a subject fordetermining presence, absence or amount of biomarker ex vivo.Non-limiting parts of the body from which a tissue sample may beobtained include leg, arm, abdomen, upper back, lower back, chest, hand,finger, fingernail, foot, toe, toenail, neck, rectum, nose, throat,mouth, scalp, face, spine, throat, heart, lung, breast, kidney, liver,intestine, colon, pancreas, bladder, cervix, testes, muscle, skin, hair,tumor or area surrounding a tumor, and the like, in some embodiments. Atissue sample can be obtained by any suitable method known in the art,including, without limitation, biopsy (e.g., shave, punch, incisional,excisional, curettage, fine needle aspirate, scoop, scallop, coreneedle, vacuum assisted, open surgical biopsies) and the like, incertain embodiments. Examples of a fluid that can be obtained from asubject includes, without limitation, blood, cerebrospinal fluid, spinalfluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal,ear, arthroscopic), urine, interstitial fluid, feces, sputum, saliva,nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile,tears, sweat, breast milk, breast fluid, fluid from region ofinflammation, fluid from region of muscle wasting and the like, in someembodiments.

A sample from a subject may be processed prior to determining presence,absence or amount of a biomarker. For example, a blood sample from asubject may be processed to yield a certain fraction, including withoutlimitation, plasma, serum, buffy coat, red blood cell layer and thelike, and biomarker presence, absence or amount can be determined in thefraction. In certain embodiments, a tissue sample (e.g., tumor biopsysample) can be processed by slicing the tissue sample and observing thesample under a microscope before and/or after the sliced sample iscontacted with an agent that visualizes a biomarker (e.g., antibody). Insome embodiments, a tissue sample can be exposed to one or more of thefollowing non-limiting conditions: washing, exposure to high salt or lowsalt solution (e.g., hypertonic, hypotonic, isotonic solution), exposureto shearing conditions (e.g., sonication, press (e.g., French press)),mincing, centrifugation, separation of cells, separation of tissue andthe like. In certain embodiments, a biomarker can be separated fromtissue and the presence, absence or amount determined in vitro. A samplealso may be stored for a period of time prior to determining thepresence, absence or amount of a biomarker (e.g., a sample may befrozen, cryopreserved, maintained in a preservation medium (e.g.,formaldehyde)).

A sample can be obtained from a subject at any suitable time ofcollection after a drug is delivered to the subject. For example, asample may be collected within about one hour after a drug is deliveredto a subject (e.g., within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 55or 60 minutes of delivering a drug), within about one day after a drugis delivered to a subject (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours ofdelivering a drug) or within about two weeks after a drug is deliveredto a subject (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13or 14 days of delivering the drug). A collection may be made on aspecified schedule including hourly, daily, semi-weekly, weekly,bi-weekly, monthly, bi-monthly, quarterly, and yearly, and the like, forexample. If a drug is administered continuously over a time period(e.g., infusion), the delay may be determined from the first moment ofdrug is introduced to the subject, from the time the drug administrationceases, or a point in-between (e.g., administration time frame midpointor other point).

Biomarker Detection

The presence, absence or amount of one or more biomarkers may bedetermined by any suitable method known in the art, and non-limitingdetermination methods are discussed herein. Determining the presence,absence or amount of a biomarker sometimes comprises use of a biologicalassay. In a biological assay, one or more signals detected in the assaycan be converted to the presence, absence or amount of a biomarker.Converting a signal detected in the assay can comprise, for example, useof a standard curve, one or more standards (e.g., internal, external), achart, a computer program that converts a signal to a presence, absenceor amount of biomarker, and the like, and combinations of the foregoing.

Biomarker detected in an assay can be full-length biomarker, a biomarkerfragment, an altered or modified biomarker (e.g., biomarker derivative,biomarker metabolite), or sum of two or more of the foregoing, forexample. Modified biomarkers often have substantial sequence identity toa biomarker discussed herein. For example, percent identity between amodified biomarker and a biomarker discussed herein may be in the rangeof 15-20%, 20-30%, 31-40%, 41-50%, 51-60%, 61-70%, 71-80%, 81-90% and91-100%, (e.g. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and100 percent identity). A modified biomarker often has a sequence (e.g.,amino acid sequence or nucleotide sequence) that is 90% or moreidentical to a sequence of a biomarker discussed herein. Percentsequence identity can be determined using alignment methods known in theart.

Detection of biomarkers may be performed using any suitable method knownin the art, including, without limitation, mass spectrometry, antibodyassay (e.g., ELISA), nucleic acid affinity, microarray hybridization,Northern blot, reverse PCR and RT-PCR. For example, RNA purity andconcentration may be determined spectrophotometrically (260/280>1.9) ona Nanodrop 1000. RNA quality may be assessed using methods known in theart (e.g., Agilent 2100 Bioanalyzer; RNA 6000 Nano LabChip® and thelike).

Indication for Adjusting or Maintaining Subsequent Drug Dose

An indication for adjusting or maintaining a subsequent drug dose can bebased on the presence or absence of a biomarker. For example, when (i)low sensitivity determinations of biomarker levels are available, (ii)biomarker levels shift sharply in response to a drug, (iii) low levelsor high levels of biomarker are present, and/or (iv) a drug is notappreciably toxic at levels of administration, presence or absence of abiomarker can be sufficient for generating an indication of adjusting ormaintaining a subsequent drug dose.

An indication for adjusting or maintaining a subsequent drug dose oftenis based on the amount or level of a biomarker. An amount of a biomarkercan be a mean, median, nominal, range, interval, maximum, minimum, orrelative amount, in some embodiments. An amount of a biomarker can beexpressed with or without a measurement error window in certainembodiments. An amount of a biomarker in some embodiments can beexpressed as a biomarker concentration, biomarker weight per unitweight, biomarker weight per unit volume, biomarker moles, biomarkermoles per unit volume, biomarker moles per unit weight, biomarker weightper unit cells, biomarker volume per unit cells, biomarker moles perunit cells and the like. Weight can be expressed as femtograms,picograms, nanograms, micrograms, milligrams and grams, for example.Volume can be expressed as femtoliters, picoliters, nanoliters,microliters, milliliters and liters, for example. Moles can be expressedin picomoles, nanomoles, micromoles, millimoles and moles, for example.In some embodiments, unit weight can be weight of subject or weight ofsample from subject, unit volume can be volume of sample from thesubject (e.g., blood sample volume) and unit cells can be per one cellor per a certain number of cells (e.g., micrograms of biomarker per 1000cells). In some embodiments, an amount of biomarker determined from onetissue or fluid can be correlated to an amount of biomarker in anotherfluid or tissue, as known in the art.

An indication for adjusting or maintaining a subsequent drug dose oftenis generated by comparing a determined level of biomarker in a subjectto a predetermined level of biomarker. A predetermined level ofbiomarker sometimes is linked to a therapeutic or efficacious amount ofdrug in a subject, sometimes is linked to a toxic level of a drug,sometimes is linked to presence of a condition, sometimes is linked to atreatment midpoint and sometimes is linked to a treatment endpoint, incertain embodiments. A predetermined level of a biomarker sometimesincludes time as an element, and in some embodiments, a threshold is atime-dependent signature. For example, an IL-6 or IL6-sR level of about8-fold more than a normal level, or greater (e.g. about 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, or 75-fold more than a normal level) mayindicate that the dosage of the drug or the frequency of administrationmay be increased in a subsequent administration.

The term “dosage” is meant to include both the amount of the dose andthe frequency of administration, such as, for example, the timing of thenext dose. An IL-6 or IL-6sR level less than about 8-fold more than anormal level (e.g. about 7, 6, 5, 4, 3, 2, or 1-fold more than a normallevel, or less than or equal to a normal level) may indicate that thedosage may be maintained or decreased in a subsequent administration. AVCAM-1 level of about 8 fold more than a normal level, or greater (e.g.e.g. about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75-foldmore than a normal level) may indicate that the dosage of the drug maybe increased in a subsequent administration. A VCAM-1 level less thanabout 8-fold more than a normal level (e.g. about 7, 6, 5, 4, 3, 2, or1-fold more than a normal level, or less than or equal to a normallevel) may indicate that the dosage may be maintained or decreased in asubsequent administration. A normal level of IL-6, IL-6sR, or VCAM-1 maybe assessed in a subject not diagnosed with a solid tumor or the type ofsolid tumor under treatment in a patient.

Other indications for adjusting or maintaining a drug dose include, forexample, a perturbation in the concentration of an individual secretedfactor, such as, for example, GM-CSF, MIP-1α, MIP-1β, MCP-1, IFN-γ,RANTES, EGF or HGF, or a perturbation in the mean concentration of apanel of secreted factors, such as two or more of the markers selectedfrom the group consisting of GM-CSF, MIP-1α, MIP-1β, MCP-1, IFN-γ,RANTES, EGF and HGF. This perturbation may, for example, consist of anincrease, or decrease, in the concentration of an individual secretedfactor by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% or an increase or decrease in the mean relative change in serumconcentration of a panel of secreted factors by at least 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. This increase may, or maynot, be followed by a return to baseline serum concentrations before thenext administration. The increase or decrease in the mean relativechange in serum concentration may involve, for example, weighting therelative value of each of the factors in the panel. Also, the increaseor decrease may involve, for example, weighting the relative value ofeach of the time points of collected data. The weighted value for eachtime point or each factor may vary, depending on the state or the extentof the cancer, metastasis, or tumor burden. An indication for adjustingor maintaining the drug dose may include a perturbation in theconcentration of an individual secreted factor or the mean concentrationof a panel of secreted factors, after 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10or more administrations. For example, where it is observed that over thecourse of treatment, for example, 6 administrations of a drug or thevaccines or compositions discussed herein, that the concentration of anindividual secreted factor or the mean concentration of a panel ofsecreted factors is perturbed after at least one administration, thenthis may be an indication to maintain, decrease, or increase thefrequency of administration or the subsequent dosage, or it may be anindication to continue treatment by, for example, preparing additionaldrug, adenovirus vaccine, or adenovirus transfected or transduced cells.

Some treatment methods comprise (i) administering a drug to a subject inone or more administrations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10doses), (ii) determining the presence, absence or amount of a biomarkerin or from the subject after (i), (iii) providing an indication ofincreasing, decreasing or maintaining a subsequent dose of the drug foradministration to the subject, and (iv) optionally administering thesubsequent dose to the subject, where the subsequent dose is increased,decreased or maintained relative to the earlier dose(s) in (i). In someembodiments, presence, absence or amount of a biomarker is determinedafter each dose of drug has been administered to the subject, andsometimes presence, absence or amount of a biomarker is not determinedafter each dose of the drug has been administered (e.g., a biomarker isassessed after one or more of the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth or tenth dose, but not assessed every timeafter each dose is administered).

An indication for adjusting a subsequent drug dose can be considered aneed to increase or a need to decrease a subsequent drug dose. Anindication for adjusting or maintaining a subsequent drug dose can beconsidered by a clinician, and the clinician may act on the indicationin certain embodiments. In some embodiments, a clinician may opt not toact on an indication. Thus, a clinician can opt to adjust or not adjusta subsequent drug dose based on the indication provided.

An indication of adjusting or maintaining a subsequent drug dose, and/orthe subsequent drug dosage, can be provided in any convenient manner. Anindication may be provided in tabular form (e.g., in a physical orelectronic medium) in some embodiments. For example, a biomarkerthreshold may be provided in a table, and a clinician may compare thepresence, absence or amount of the biomarker determined for a subject tothe threshold. The clinician then can identify from the table anindication for subsequent drug dose. In certain embodiments, anindication can be presented (e.g., displayed) by a computer after thepresence, absence or amount of a biomarker is provided to computer(e.g., entered into memory on the computer). For example, presence,absence or amount of a biomarker determined for a subject can beprovided to a computer (e.g., entered into computer memory by a user ortransmitted to a computer via a remote device in a computer network),and software in the computer can generate an indication for adjusting ormaintaining a subsequent drug dose, and/or provide the subsequent drugdose amount. A subsequent dose can be determined based on certainfactors other than biomarker presence, absence or amount, such as weightof the subject, one or more metabolite levels for the subject (e.g.,metabolite levels pertaining to liver function) and the like, forexample.

Once a subsequent dose is determined based on the indication, aclinician may administer the subsequent dose or provide instructions toadjust the dose to another person or entity. The term “clinician” asused herein refers to a decision maker, and a clinician is a medicalprofessional in certain embodiments. A decision maker can be a computeror a displayed computer program output in some embodiments, and a healthservice provider may act on the indication or subsequent drug dosedisplayed by the computer. A decision maker may administer thesubsequent dose directly (e.g., infuse the subsequent dose into thesubject) or remotely (e.g., pump parameters may be changed remotely by adecision maker).

A subject can be prescreened to determine whether or not the presence,absence or amount of a particular biomarker may be determined.Non-limiting examples of prescreens include identifying the presence orabsence of a genetic marker (e.g., polymorphism, particular nucleotidesequence); identifying the presence, absence or amount of a particularmetabolite. A prescreen result can be used by a clinician in combinationwith the presence, absence or amount of a biomarker to determine whethera subsequent drug dose may be adjusted or maintained.

Biomarkers for assessing the effect of the modified T cells and nucleicacids herein may include, for example, IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17, IL-25, IFN-γ, TNF-α, TNFβ, GM-CSF, TGFβ, C-reactiveprotein and others.

Antibodies and Small Molecules

In some embodiments, an antibody or small molecule is provided for useas a control or standard in an assay, or a therapeutic, for example. Insome embodiments, an antibody or other small molecule configured to bindto a cytokine or cytokine receptor, including without limitation IL-6,IL-6sR, and alter the action of the cytokine, or it may be configured tobind to VCAM-1. In certain embodiments an antibody or other smallmolecule may bind to an mRNA structure encoding for a cytokine orreceptor.

The term small molecule as used herein means an organic molecule ofapproximately 800 or fewer Daltons. In certain embodiments smallmolecules may diffuse across cell membranes to reach intercellular sitesof action. In some embodiments a small molecule binds with high affinityto a biopolymer such as protein, nucleic acid, or polysaccharide and maysometimes alter the activity or function of the biopolymer. In variousembodiments small molecules may be natural (such as secondarymetabolites) or artificial (such as antiviral drugs); they may have abeneficial effect against a disease (such as drugs) or may bedetrimental (such as teratogens and carcinogens). By way of non-limitingexample, small molecules may include ribo- or deoxyribonucleotides,amino acids, monosaccharides and small oligomers such as dinucleotides,peptides such as the antioxidant glutathione, and disaccharides such assucrose.

The term antibody as used herein is to be understood as meaning a gammaglobulin protein found in blood or other bodily fluids of vertebrates,and used by the immune system to identify and neutralize foreignobjects, such as bacteria and viruses. Antibodies typically includebasic structural units of two large heavy chains and two small lightchains.

Specific binding to an antibody requires an antibody that is selectedfor its affinity for a particular protein. For example, polyclonalantibodies raised to a particular protein, polymorphic variants,alleles, orthologs, and conservatively modified variants, or splicevariants, or portions thereof, can be selected to obtain only thosepolyclonal antibodies that are specifically immunoreactive with GM-CSF,TNF-α or NF-κ-B modulating protein and not with other proteins. Thisselection may be achieved by subtracting out antibodies that cross-reactwith other molecules.

Methods as presented herein include without limitation the delivery ofan effective amount of an activated cell, a nucleic acid, or anexpression construct encoding the same. An “effective amount” of thepharmaceutical composition, generally, is defined as that amountsufficient to detectably and repeatedly to achieve the stated desiredresult, for example, to ameliorate, reduce, minimize or limit the extentof the disease or its symptoms. Other more rigorous definitions mayapply, including elimination, eradication or cure of disease. In someembodiments there may be a step of monitoring the biomarkers to evaluatethe effectiveness of treatment and to control toxicity.

An effective amount of the modified cell may be determined by aphysician, considering the individual patient. Factors to be consideredmay include, for example, the extent of the disease or condition, tumorsize, extent of infection, metastasis, age, and weight. The dosage andnumber of administrations may be determined by the physician, or otherclinician, by monitoring the patient for disease or condition symptoms,and for responses to previous dosages, for example, by monitoring tumorsize, or the level or concentration of tumor antigen. In certainexamples, the modified cells may be administered at a dosage of 10⁴ to10⁹ modified cells/kg body weight, 10⁵ to 10⁶, 10⁹-10¹¹, or 10¹⁰-10¹¹modified cells/kg body weight.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology. Examples herein that discuss the methods fortransforming or transfecting cells in vitro, or ex vivo, provideexamples of, but do not limit, the use of nucleic acids that expresschimeric polypeptides. Examples of the delivery of the transfected ortransduced cells, and ligand inducer, to laboratory animals or humansubjects provide examples of, but do not limit, the directadministration of nucleic acids expressing chimeric polypeptides, tumorantigens, and ligand inducer to subjects in need thereof.

Expression of Inducible Chimeric MyD88/CD40 Polypeptides

Examples 1-7 relate to the expression of an inducible form of chimericstimulating molecules and provide examples of methods that may be usedto express the MyD88/CD40 chimeric stimulating molecules discussedherein. These examples may be used as a reference for constructingassaying, and using the chimeric stimulating molecules, and chimericantigen receptors discussed herein. Examples 8 et seq. relate to theexpression and applications of the non-inducible MyD88/CD40 chimericstimulating molecules and the MyD88/CD40 chimeric antigen receptorsdiscussed herein.

Example 1: Inducible Chimeric Stimulating Molecules

Inducible MyD88/CD40 chimeric costimulatory molecules were expressed inT cells; contacting the T cells with rimiducid (AP1903) resulted inactivation of costimulatory activity and activation of T cells,including T cells that co-expressed a chimeric antigen receptor.

The following patents, applications, and patent publications may containmaterial and methods that may be used in the examples herein, such as,for example, U.S. Pat. No. 7,404,950, issued Jul. 29, 2008, to Spencer,D. et al.; U.S. Pat. No. 8,691,210, issued Apr. 8 2004 to Spencer, etal.; U.S. patent application Ser. No. 12/532,196 by Spencer et al.,filed Sep. 21, 2009; PCT application PCT/US2009/057738 to Spencer etal., published on Apr. 24, 2008 as WO2010/033949; U.S. patentapplication Ser. No. 13/087,329 by Slawin et al., filed Apr. 14, 2011;PCT application PCT/US2011/032572, published on Oct. 20, 2011 asWO2011/130566; U.S. patent application Ser. No. 14/210,324 by Spencer etal., filed Mar. 13, 2014; PCT application number PCT/US2014/026734 bySpencer et al., published as WO2014/251960 on Feb. 5, 2015; U.S.application Ser. No. 14/622,018, by Foster et al., filed Feb. 13, 2015;PCT application number PCT/US2015/015829 by Foster et al., published asWO2015/123527 on Aug. 20, 2015 are all hereby incorporated by referenceherein in their entirety.

Examples of adenoviral vectors used for expression of an inducibleMyD88/CD40 chimeric stimulating molecule are provided herein. Thesevectors may be modified to remove the FKBP regions to obtain adenoviralvectors that express non-inducible chimeric stimulating molecules of thepresent application.

The following nucleotide sequences were used to construct theAd5-iMC-P2A-P-FL and Ad5f35-iMC-P2A-P-FL. vectors. The amino acidsequences of the polypeptides coded by the nucleotide sequences are alsoprovided.

Ad-iMC-2A-P-FL SEQ ID NO: 1 Myratggggagtagcaagagcaagcctaaggaccccagccagcgc SEQ ID NO: 2 MyrMGSSKSKPKDPSQR SEQ ID NO: 3 MyD88atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgatctgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgccttcatctgcta ttgccccagcgacatcSEQ ID NO: 4 MyD88 MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 5 CD40aaaaaggtggccaagaagccaaccaataaggccccccaccccaagcaggagccccaggagatcaattttcccgacgatcttcctggctccaacactgctgctccagtgcaggagactttacatggatgccaaccggtcacccaggaggatggcaaagagagtcgcatctcagtgcaggagagacag SEQ ID NO: 6 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQED GKESRISVQERQSEQ ID NO: 7 Fv′ GGcGTcCAaGTcGAaACcATtagtCCcGGcGAtGGcaGaACaTTtCCtAAaaGgGGaCAaACaTGtGTcGTcCAtTAtACaGGcATGtTgGAgGAcGGcAAaAAgGTgGAcagtagtaGaGAtcGcAAtAAaCCtTTcAAaTTcATGtTgGGaAAaCAaGAaGTcATtaGgGGaTGGGAgGAgGGcGTgGCtCAaATGtccGTcGGcCAacGcGCtAAgCTcACcATcagcCCcGAcTAcGCaTAcGGcGCtACcGGaCAtCCcGGaATtATtCCcCCtCAcGCtACctTgGTgTTtGAc GTcGAaCTgtTgAAgCTcSEQ ID NO: 8: Fv′ GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFD VELLKLSEQ ID NO: 9 Fv ggagtgcaggtggagactatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaagttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgat gtggagcttctaaaactggaaSEQ ID NO: 10 Fv GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFD VELLKLESEQ ID NO: 11 P2A GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT SEQ ID NO: 12 P2A ATNFSLLKQAGDVEENPGP SEQ ID NO: 13 PSMAATGTGGAATCTCCTTCACGAAACCGACTCGGCTGTGGCCACCGCGCGCCGCCCGCGCTGGCTGTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTCGGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATGAAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTACATAATTTTACACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTCAATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTAGCACATTATGATGTCCTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGATGGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAATGTTTGGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAgAGGGCGATCTAGTGTATGTTaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgctgagactttgagtgaagtagcc taaSEQ ID NO: 14 PSMA MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLHNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVWDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA

pAd1127-02.iMC-P2A-P-FL is the shuttle vector used to make bothAd5-iMC-P2A-P-FL and the serotype 35 pseudotyped Ad5f35-iMC-P2A-P-FL. Itcontains the inducible MyD88/CD40 and full length PSMA on the sametranscript driven by a CMVp and bovine growth hormone poly A site.

Annotated Vector Sequence:

8931 bp ds-DNA /note = “MMLV Psi”/note = “packaging signal of Moloney murine leukemia virus (MMLV)” CDS1226 . . . 3802 /codon_start = 1 /note = “iMC-2A-Delta-CD19”/translation = ” SEQ ID NO: 15MGSSKSKPKDPSQRLEMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQVESGGGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEVEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDRAKRGKPIPNPLLGLDSTGSGSATNFSLLKQAGDVEENPGPTRMPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF” misc_feature1226 . . . 1267 /note = “Myr” misc_feature 1268 . . . 1273/note = “XhoI” misc_feature 1274 . . . 1789 /note = “MyD88” misc_feature1790 . . . 1975 /note = “Delta-CD40” misc_feature 1976 /note = “SalI”misc_feature 1977 . . . 1981 /note = “XhoI” misc_feature 1982 . . . 1999/note = “L1” misc_feature 2000 . . . 2320 /note = “LFv1” misc_feature2327 . . . 2647 /note = “Fv2L” misc_feature 2648 . . . 2665 /note = “L1”misc_feature 2666 /note = “SalI” misc_feature 2667 . . . 2671/note = “SalI” misc_feature 2672 . . . 2683 /note = “Furin” misc_feature2684 . . . 2725 /product = “epitope tag from simian virus 5”/note = “V5 tag” misc_feature 2726 . . . 2794 /note = “L-2A”misc_feature 2795 . . . 2800 /note = “MluI” misc_feature 2801 . . . 3802/note = “dCD19” misc_feature 3962 . . . 4551 /note = “LTR” primer_bindcomplement(5250 . . . 5266) /note = “M13 fwd”/note = “common sequencing primer, one of multiple similar variants”promoter 5741 . . . 5845 /gene = “bla” /note = “AmpR promoter” CDS5846 . . . 6706 /codon_start = 1 /gene = “bla”/product = “beta-lactamase” /note = “AmpR”/note = “confers resistance to ampicillin, carbenicillin, andrelated antibiotics” /translation = ” SEQ ID NO: 16MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW”rep_origin 6877 . . . 7465 /direction = RIGHT /note = “ori”/note = “high-copy-number colE1/pMB1/pBR322/pUC origin of replication”promoter 7789 . . . 7819 /note = “lac promoter”/note = “promoter for the E. coli lac operon” protein_bind7827 . . . 7843 /bound_moiety = “lac repressor encoded by lacI”/note = “lac operator”/note = “The lac repressor binds to the lac operator toinhibit transcription in E. coli. This inhibition can berelieved by adding lactose orisopropyl-β-D-thiogalactopyranoside (IPTG).” primer_bind 7851 . . . 7867/note = “M13 rev”/note = “common sequencing primer, one of multiple similar variants” LTR8276 . . . 8869/note = “long terminal repeat from Moloney murine leukemia virus”SEQ ID NO: 17 ORIGIN 1aagctggcca gcaacttatc tgtgtctgtc cgattgtcta gtgtctatga ctgattttat 61gcgcctgcgt cggtactagt tagctaacta gctctgtatc tggcggaccc gtggtggaac 121tgacgagttc ggaacacccg gccgcaaccc tgggagacgt cccagggact tcgggggccg 181tttttgtggc ccgacctgag tcctaaaatc ccgatcgttt aggactcttt ggtgcacccc 241ccttagagga gggatatgtg gttctggtag gagacgagaa cctaaaacag ttcccgcctc 301cgtctgaatt tttgctttcg gtttgggacc gaagccgcgc cgcgcgtctt gtctgctgca 361gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa aatatgggcc 421cgggctagcc tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc 481ggatcgctca caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg 541cagaatggcc aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca 601tcacccaggt taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtgg 661ggtacatcgt gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg 721tacaccctaa gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc 781ctcgttcgac cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccc 841ccatatggcc atatgagatc ttatatgggg cacccccgcc ccttgtaaac ttccctgacc 901ctgacatgac aagagttact aacagcccct ctctccaagc tcacttacag gctctctact 961tagtccagca cgaagtctgg agacctctgg cggcagccta ccaagaacaa ctggaccgac 1021cggtggtacc tcacccttac cgagtcggcg acacagtgtg ggtccgccga caccagacta 1081agaacctaga acctcgctgg aaaggacctt acacagtcct gctgaccacc cccaccgccc 1141tcaaagtaga cggcatcgca gcttggatac acgccgccca cgtgaaggct gccgaccccg 1201ggggtggacc atcctctaga ctgccatggg gagtagcaag agcaagccta aggaccccag 1261ccagcgcctc gagatggccg ctgggggccc aggcgccgga tcagctgctc ccgtatcttc 1321tacttcttct ttgccgctgg ctgctctgaa catgcgcgtg agaagacgcc tctccctgtt 1381ccttaacgtt cgcacacaag tcgctgccga ttggaccgcc cttgccgaag aaatggactt 1441tgaatacctg gaaattagac aacttgaaac acaggccgac cccactggca gactcctgga 1501cgcatggcag ggaagacctg gtgcaagcgt tggacggctc ctggatctcc tgacaaaact 1561gggacgcgac gacgtactgc ttgaactcgg acctagcatt gaagaagact gccaaaaata 1621tatcctgaaa caacaacaag aagaagccga aaaacctctc caagtcgcag cagtggactc 1681atcagtaccc cgaacagctg agcttgctgg gattactaca ctcgacgacc cactcggaca 1741tatgcctgaa agattcgacg ctttcatttg ctattgcccc tctgacataa agaaagttgc 1801aaagaaaccc acaaataaag ccccacaccc taaacaggaa ccccaagaaa tcaatttccc 1861agatgatctc cctggatcta atactgccgc cccggtccaa gaaaccctgc atggttgcca 1921gcctgtcacc caagaggacg gaaaagaatc acggattagc gtacaagaga gacaagtcga 1981gtctggcggt ggatccggag gcgttcaagt agaaacaatc agcccaggag acggaaggac 2041tttccccaaa cgaggccaaa catgcgtagt tcattatact gggatgctcg aagatggaaa 2101aaaagtagat agtagtagag accgaaacaa accatttaaa tttatgttgg gaaaacaaga 2161agtaataagg ggctgggaag aaggtgtagc acaaatgtct gttggccagc gcgcaaaact 2221cacaatttct cctgattatg cttacggagc taccggccac cccggcatca taccccctca 2281tgccacactg gtgtttgacg tcgaattgct caaactggaa gtcgagggag tgcaggtgga 2341gacgattagt cctggggatg ggagaacctt tccaaagcgc ggtcagacct gtgttgtcca 2401ctacaccggt atgctggagg acgggaagaa ggtggactct tcacgcgatc gcaataagcc 2461tttcaagttc atgctcggca agcaggaggt gatccggggg tgggaggagg gcgtggctca 2521gatgtcggtc gggcaacgag cgaagcttac catctcaccc gactacgcgt atggggcaac 2581ggggcatccg ggaattatcc ctccccacgc tacgctcgta ttcgatgtgg agctcttgaa 2641gcttgagtct ggcggtggat ccggagtcga ccgcgcaaag cgtggaaaac ctatacctaa 2701tccattgctg ggcttagact caacaggcag cggaagcgca acgaattttt ccctgctgaa 2761acaggcaggg gacgtagagg aaaatcctgg tcctacgcgt atgccccctc ctagactgct 2821gtttttcctg ctctttctca ccccaatgga agttagacct gaggaaccac tggtcgttaa 2881agtggaagaa ggtgataatg ctgtcctcca atgccttaaa gggaccagcg acggaccaac 2941gcagcaactg acttggagcc gggagtcccc tctcaagccg tttctcaagc tgtcacttgg 3001cctgccaggt cttggtattc acatgcgccc ccttgccatt tggctcttca tattcaatgt 3061gtctcaacaa atgggtggat tctacctttg ccagcccggc cccccttctg agaaagcttg 3121gcagcctgga tggaccgtca atgttgaagg ctccggtgag ctgtttagat ggaatgtgag 3181cgaccttggc ggactcggtt gcggactgaa aaataggagc tctgaaggac cctcttctcc 3241ctccggtaag ttgatgtcac ctaagctgta cgtgtgggcc aaggaccgcc ccgaaatctg 3301ggagggcgag cctccatgcc tgccgcctcg cgattcactg aaccagtctc tgtcccagga 3361tctcactatg gcgcccggat ctactctttg gctgtcttgc ggcgttcccc cagatagcgt 3421gtcaagagga cctctgagct ggacccacgt acaccctaag ggccctaaga gcttgttgag 3481cctggaactg aaggacgaca gacccgcacg cgatatgtgg gtaatggaga ccggccttct 3541gctccctcgc gctaccgcac aggatgcagg gaaatactac tgtcatagag ggaatctgac 3601tatgagcttt catctcgaaa ttacagcacg gcccgttctt tggcattggc tcctccggac 3661tggaggctgg aaggtgtctg ccgtaacact cgcttacttg attttttgcc tgtgtagcct 3721ggttgggatc ctgcatcttc agcgagccct tgtattgcgc cgaaaaagaa aacgaatgac 3781tgaccctaca cgacgattct gagcatgcaa cctcgatccg gattagtcca atttgttaaa 3841gacaggatat cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct 3901atagagtacg agccatagat aaaataaaag attttattta gtctccagaa aaagggggga 3961atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca 4021tggaaaaata cataactgag aatagagaag ttcagatcaa ggtcaggaac agatggaaca 4081gctgaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca 4141agaacagatg gaacagctga atatgggcca aacaggatat ctgtggtaag cagttcctgc 4201cccggctcag ggccaagaac agatggtccc cagatgcggt ccagccctca gcagtttcta 4261gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt 4321gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa 4381taaaagagcc cacaacccct cactcggggc gccagtcctc cgattgactg agtcgcccgg 4441gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc 4501ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc acacatgcag 4561catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg gccatagtac 4621ttaaagttac attggcttcc ttgaaataaa catggagtat tcagaatgtg tcataaatat 4681ttctaatttt aagatagtat ctccattggc tttctacttt ttcttttatt tttttttgtc 4741ctctgtcttc catttgttgt tgttgttgtt tgtttgtttg tttgttggtt ggttggttaa 4801ttttttttta aagatcctac actatagttc aagctagact attagctact ctgtaaccca 4861gggtgacctt gaagtcatgg gtagcctgct gttttagcct tcccacatct aagattacag 4921gtatgagcta tcatttttgg tatattgatt gattgattga ttgatgtgtg tgtgtgtgat 4981tgtgtttgtg tgtgtgactg tgaaaatgtg tgtatgggtg tgtgtgaatg tgtgtatgta 5041tgtgtgtgtg tgagtgtgtg tgtgtgtgtg tgcatgtgtg tgtgtgtgac tgtgtctatg 5101tgtatgactg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgttgtga 5161aaaaatattc tatggtagtg agagccaacg ctccggctca ggtgtcaggt tggtttttga 5221gacagagtct ttcacttagc ttggaattca ctggccgtcg ttttacaacg tcgtgactgg 5281gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 5341cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 5401gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 5461tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 5521ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 5581gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 5641gcgatgacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 5701ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 5761atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 5821tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 5881cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 5941agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 6001taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 6061tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 6121catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 6181ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 6241ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 6301catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 6361aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6421aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 6481taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6541atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 6601gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 6661tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 6721ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 6781gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 6841agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 6901aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 6961agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 7021tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 7081atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 7141taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 7201gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 7261gcgtgagcat tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 7321aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 7381tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 7441gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 7501cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 7561ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 7621cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 7681ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 7741gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 7801gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 7861ctatgaccat gattacgcca agctttgctc ttaggagttt cctaatacat cccaaactca 7921aatatataaa gcatttgact tgttctatgc cctagggggc ggggggaagc taagccagct 7981ttttttaaca tttaaaatgt taattccatt ttaaatgcac agatgttttt atttcataag 8041ggtttcaatg tgcatgaatg ctgcaatatt cctgttacca aagctagtat aaataaaaat 8101agataaacgt ggaaattact tagagtttct gtcattaacg tttccttcct cagttgacaa 8161cataaatgcg ctgctgagca agccagtttg catctgtcag gatcaatttc ccattatgcc 8221agtcatatta attactagtc aattagttga tttttatttt tgacatatac atgtgaatga 8281aagaccccac ctgtaggttt ggcaagctag cttaagtaac gccattttgc aaggcatgga 8341aaaatacata actgagaata gaaaagttca gatcaaggtc aggaacagat ggaacagctg 8401aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa 8461cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg 8521gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag tttctagaga 8581accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac 8641taaccaatca gttcgcttct cgcttctgtt cgcgcgctta tgctccccga gctcaataaa 8701agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac 8761ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg 8821gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt 8881ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg t

Example 2: Using the Inducible CSM in Human Cells for Therapy

Presented in this example are expression constructs and methods of usingthe expression constructs in human cells. Although this example refersto the inducible chimeric stimulating molecule, vectors coding for thenon-inducible chimeric stimulating molecules, and the MyD88/CD40chimeric antigen receptors may also be used, including any appropriatemodifications.

These methods may be adapted for other cells, such as, for example NKand NKT cells, as well as tumor-infiltrating lymphocytes, and may alsobe adapted for chimeric stimulating polypeptides that comprise othercostimulatory polypeptide cytoplasmic regions as discussed herein.

Materials and Methods

Large-Scale Generation of Gene-Modified T Cells

T cells are generated from healthy volunteers, using standard methods.Briefly, peripheral blood mononuclear cells (PBMCs) from healthy donorsor cancer patients are activated for expansion and transduction usingsoluble αCD3ζ and αCD28 (Miltenyi Biotec, Auburn, Calif.). PBMCs areresuspended in Cellgenix DC media supplemented with 100 U/ml IL-2(Cellgenix) at 1×10⁶ cells/ml and stimulated with 0.2 μg/ml αCD3ζ and0.5 μg/ml αCD28 soluble antibody. Cells are then cultured at 37° C., 5%CO₂ for 4 days. On day four, 1 ml of fresh media containing IL-2 isadded. On day 7, cells are harvested and resuspended in Cellgenix DCmedia for transduction.

Plasmid and Retrovirus

The SFG plasmid consists of inducible CSM linked, via a cleavable2A-like sequence, to truncated human CD19. The inducible CSM consists ofa human FK506-binding protein (FKBP12; GenBank AH002 818) with an F36Vmutation, connected via a Ser-Gly-Gly-Gly-Ser linker to a human CSM. TheF36V mutation increases the binding affinity of FKBP12 to the synthetichomodimerizer, AP20187 or AP1903. The 2A-like sequence encodes a 20amino acid peptide from Thosea asigna insect virus, which mediates >95%cleavage between a glycine and terminal proline residue, resulting in 19extra amino acids in the C terminus of iCSM, and one extra prolineresidue in the N terminus of CD19. CD19 consists of full-length CD19(GenBank NM 001770) truncated at amino acid 333 (TDPTRRF), whichshortens the intracytoplasmic domain from 242 to 19 amino acids, andremoves all conserved tyrosine residues that are potential sites forphosphorylation.

A stable PG13-based clone producing Gibbon ape leukemia virus (Gal-V)pseudotyped retrovirus is made by transiently transfecting Phoenix Ecocell line (ATCC product # SD3444; ATCC, Manassas, Va.) with the SFGplasmid. This produces Eco-pseudotyped retrovirus. The PG13 packagingcell line (ATCC) is transduced three times with Eco-pseudotypedretrovirus to generate a producer line that contained multiple SFGplasmid proviral integrants per cell. Single cell cloning is performed,and the PG13 clone that produced the highest titer is expanded and usedfor vector production.

Retroviral Transduction

Culture medium for T cell activation and expansion is serum-freeCellgenix DC medium (Cellgenix) supplemented by 100 U/ml IL-2(Cellgenix). T cells are activated by soluble anti-CD3 and anti-CD28(Miltenyi Biotec) for 7 days before transduction with retroviral vector.Immunomagnetic selection of ΔCD19, if necessary, is performed on day 4after transduction; the positive fraction was expanded for a further 2days and cryopreserved.

Scaling-Up Production of Gene-Modified Allodepleted Cells

Scale-up of the transduction process for clinical application usenon-tissue culture-treated T75 flasks (Nunc, Rochester, N.Y.), which arecoated with 10 ml of anti-CD3 0.5 μg/ml and anti-CD28 0.2 μg/ml or 10 mlof fibronectin 7 μg/ml at 4° C. overnight. Fluorinated ethylenepropylene bags corona-treated for increased cell adherence (2PF-0072AC,American Fluoroseal Corporation, Gaithersburg, Md.) are also used. PBMCsare seeded in anti-CD3, anti-CD28-coated flasks at 1×10⁶ cells/ml inmedia supplemented with 100 U/ml IL-2. For retroviral transduction,retronectin-coated flasks or bags are loaded once with 10 ml ofretrovirus-containing supernatant for 2 to 3 hours. Activated T cellsare seeded at 1×10⁶ cells/ml in fresh retroviral vector-containingmedium and T cell culture medium at a ratio of 3:1, supplemented with100 U/ml IL-2. Cells are harvested the following morning and expanded intissue-culture treated T75 or T175 flasks in culture medium supplementedwith 100 U/ml IL-2 at a seeding density of between about 5×10⁵ cells/mlto 8×10⁵ cells/ml.

CD19 Immunomagnetic Selection

Immunomagnetic selection for CD19 may, for example, be performed 4 daysafter transduction. Cells are labeled with paramagnetic microbeadsconjugated to monoclonal mouse anti-human CD19 antibodies (MiltenyiBiotech, Auburn, Calif.) and selected on MS or LS columns in small scaleexperiments and on a CliniMacs Plus automated selection device in largescale experiments. CD19-selected cells are expanded for a further 4 daysand cryopreserved on day 8 post transduction. These cells are referredto as “gene-modified cells”.

Immunophenotyping and Pentamer Analysis

Flow cytometric analysis (FACSCalibur and CellQuest software; BectonDickinson) is performed using the following antibodies: CD3, CD4, CD8,CD19, CD25, CD27, CD28, CD45RA, CD45RO, CD56 and CD62L. CD19-PE (Clone4G7; Becton Dickinson) is found to give optimum staining and was used inall subsequent analysis. A non-transduced control is used to set thenegative gate for CD19. CAR expression is assessed using anti-F(ab′)2(Jackson ImmunoResearch Laboratories, West Grove, Pa.).

Statistical Analysis

Paired, 2-tailed Student's t test is used to determine the statisticalsignificance of differences between samples. All data are represented asmean±1 standard deviation.

Example 3: Treatment of a Leukemia Patient

The present example of the treatment of a leukemia patient havingadvanced treatment refractory leukemia, using the methods of the presentapplication, may also be applied to other conditions or diseases, suchas, for example, other hyperproliferative diseases or solid tumors. Themethods may be used essentially as discussed, with the understandingthat the single chain variable fragment may vary according to the targetantigen.

T cells are transduced with a nucleic acid comprising a polynucleotidecoding for a chimeric stimulating molecule of the present application.The T cells are also transduced with a nucleic acid comprising apolynucleotide coding for a chimeric antigen receptor. In otherexamples, the nucleic acid used to transduce the T cells may include,for example, a polynucleotide coding for the chimeric antigen receptor.The chimeric antigen receptor comprises a single chain variable fragmentthat recognizes CD19.

The patient undergoes lymphodepletive conditioning, followed byadministration of the transduced CD19-targeted T cells. The T cells maybe autologous, allogeneic, or non-allogeneic. The dose may be provided,for example, daily, twice a week, or weekly. Because of the concern thatan unregulated, too rapid rate of T cell expansion, activation, andtumor cell killing may lead to a more severe cytokine storm thatunnecessarily harms the patient, the dosing schedule is designed toachieve a complete recovery at a rate that limits toxicity and does notcause extensive harm to the patient, for example, keeping the patientout of the intensive care unit at a hospital.

Example 4: Measurement of iMC Activity in CAR-Transduced T Cells

This Example discusses the use of an inducible chimeric stimulatorymolecule, but methods discussed herein may also applied to anon-inducible chimeric stimulatory molecule.

Aim: To transduce primary T cells with a retroviral vector encodingsignaling molecules linked to two FKBPv36 molecules to allow AP1903activation of the T cells. The experiment is designed to examine whetherthe inducible costimulatory molecule comprising the truncated MyD88 andCD40 polypeptides, improve killing of the GFP-modified Capan-1(pancreatic adenocarcinoma) cells by T cells also transduced with a CARrecognizing prostate stem cell antigen (PSCA), which is highly expressedon Capan-1 tumor cells.

Methods: Design and Cloning of Inducible T Cell Molecules:

1. Transduction of T cells is performed with RV-172(SFG-Myr.MyD88/CD40.Fv.Fv′.2A.ΔCD19) and RV-89(SFG.PSCAscFv.CH₂CH₃.CD28.ζ). The scFv targets PSCA using the scFv fromthe humanized monoclonal antibody, 1G8 (derived from humanized anti-PSCAin US2012077962 A1). This is linked to the CH₂CH₃ region of human IgG1,which in turn is linked to CD28 which contains both the transmembraneand cytoplasmic portion of the molecule. CD28 is linked to thecytoplasmic portion of CD3ζ.

Production of Retrovirus:

2. Essentially the same as in the previous example.

Generation of GFP-Marked Capan-1 (Pancreatic Adenocarcinoma) Cell Line:

3. Capan-1 is purchased from ATCC. Subsequently, the cell line isgene-modified by transfection with the pBP0168-pcDNA3.1-EGFPluc whichcontains the gene for the EGFP/firefly luciferase fusion protein, aswell as the neomycin resistance gene allowing stably transfected cellsto be selected over time by culturing with G418 antibiotic. Followingculture, clones with high GFP expression are selected and subcultureduntil a cell line with >95% GFP is obtained.Co-Culture of iMC-Enabled T Cells with Capan-1 Tumor Cells:4. Non-transduced or T cells co-transduced with RV-89 (PSCA CAR) andRV-172 (iMC vector) are cultured at a 5:1 ratio of T cells to tumorcells in media supplemented with 50 U/ml IL-2, and either with orwithout 10 nM AP1903. Co-cultures are then incubated at 37° C. and 5%CO₂ for 72 hours. Cultures are subsequently analyzed for the presence ofGFP⁺ tumor cells by fluorescent microscopy and by harvesting thecultures with 0.25% trypsin/EDTA and measuring the frequency of GFP⁺CD3⁻tumor cells in the culture by flow cytometry.

Results:

1. The cultures are examined by fluorescent microscopy to assess animprovement in tumor cell killing in the wells that contain theinducible costimulating molecule- and chimeric antigenreceptor-transduced T cells and that received AP1903.2. Flow cytometry is used to analyze GFP⁺ cells in the culturesfollowing trypsinization to determine whether AP1903 contributes to areduction in tumor cell number in this short culture period (72 hours).The time period for the culture may be extended to approximately 5 days.The flow cytometry plots may show the reduction in GFP⁺ cells in wells,at a 5:1 ratio, that were transduced with both virus and receive AP1903.3. The remaining viable Capan-1-GFP cells are normalized to theconditions of NT T cells without AP1903 to show the effect of iMCactivation on tumor cell killing.

Example 5: Examples of Particular Nucleic Acid and Amino Acid Sequences

The following sequences may be used in the design of expression vectorsthat encode the chimeric antigen receptors or chimeric stimulatingmolecules provided herein.

SEQ ID NO: 18, CD28 ntTTCTGGGTACTGGTTGTAGTCGGTGGCGTACTTGCTTGTTATTCTCTTCTTGTTACCGTAGCCTTCATTATATTCTGGGTCCGATCAAAGCGCTCAAGACTCCTCCATTCCGATTATATGAACATGACACCTCGCCGACCTGGTCCTACACGCAAACATTATCAACCCTACGCACCCCCCCGAGACTTCGCTGCTTATCG ATCCSEQ ID NO: 19, CD28 aaFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSSEQ ID NO: 20, Linker sequence (between CD28 and 4-1BB) nt GGATCCSEQ ID NO: 21, Linker sequence (between CD28 and 4-1BB) aa GSSEQ ID NO: 22, 4-1BB ntAGTGTAGTTAAAAGAGGAAGAAAAAAGTTGCTGTATATATTTAAACAACCATTTATGAGACCAGTGCAAACCACCCAAGAAGAAGACGGATGTTCATGCAGATTCCCAGAAGAAGAAGAAGGAGGATGTGAATTG SEQ ID NO: 23, 4-1BB aaSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSEQ ID NO: 24, Linker sequence (between 4-1BB and CD3 ζ) nt ACGCGTSEQ ID NO: 25, Linker sequence (between 4-1BB and CD3 ζ) aa TRSEQ ID NO: 26, CD3 ζ ntCGGGTCAAATTCAGCCGGAGTGCTGACGCCCCAGCATACCAACAGGGACAAAACCAACTCTACAACGAGCTCAACCTGGGTAGACGCGAGGAGTACGACGTTCTGGATAAGAGGCGGGGCCGGGACCCAGAGATGGGGGGCAAACCTCAGCGGCGGAAGAACCCGCAGGAGGGTCTTTATAACGAGCTCCAGAAGGACAAGATGGCGGAAGCCTATTCAGAAATTGGGATGAAAGGCGAGAGACGCAGGGGAAAAGGTCACGATGGTCTGTATCAAGGACTGTCAACCGCCACCAAAGACACTTACGATGCGCTCCACATGCAGGCCCTCCCTCCCCGC SEQ ID NO: 27, CD3 ζ aaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR

The following is an example of the nucleotide and amino acid sequencesfor a chimeric antigen receptor (CAR) sequences (in order, without scFvfragments)

SEQ ID NO: 28, Signal peptide ntATGGAGTTTGGGCTGTCATGGCTGTTCCTCGTGGCCATTCTCAAAGGGGT CCAGTGTTCTCGCSEQ ID NO: 29, Signal peptide aa MGFGLSWLFLVAILKGVQCSRSEQ ID NO: 30, Flexible linker sequence ntGGGGGAGGAGGTTCTGGAGGCGGCGGGAGCGGAGGAGGAGGCAGCSEQ ID NO: 31, Flexible linker sequence aa GGGGSGGGGSGGGGSSEQ ID NO: 32, Linker sequence (between scFv and CH₂CH₃) nt GGATCCSEQ ID NO: 33, Linker sequence (between scFv and CH₂CH₃) aa GSSEQ ID NO: 34, IgG1 CH₂CH₃ ntGATCCAGCCGAACCCAAATCCCCCGATAAAACACATACTTGCCCCCCTTGTCCCGCACCAGAATTGCTTGGCGGACCTTCCGTTTTTCTTTTTCCCCCCAAACCTAAAGATACCCTGATGATTTCCCGAACCCCTGAAGTTACGTGCGTAGTCGTAGATGTGTCTCACGAAGATCCAGAAGTAAAATTTAACTGGTACGTAGATGGAGTCGAAGTTCACAACGCAAAGACGAAGCCCCGAGAAGAACAATATAATTCCACATACCGAGTAGTTAGCGTTCTCACCGTACTGCATCAGGACTGGCTTAACGGCAAAGAATATAAATGTAAGGTCTCAAACAAAGCACTCCCAGCCCCTATCGAAAAGACTATCTCCAAAGCTAAAGGACAACCCCGCGAACCCCAGGTCTATACACTTCCCCCCTCACGCGATGAACTCACTAAAAATCAGGTTTCCCTTACTTGTCTTGTCAAAGGCTTCTACCCTAGCGATATCGCAGTCGAATGGGAATCCAATGGCCAGCCCGAAAACAACTATAAAACAACCCCACCTGTCCTCGATTCAGATGGCTCATTCTTTCTCTATTCCAAACTGACTGTAGACAAATCCCGATGGCAACAAGGTAACGTGTTCTCTTGCTCAGTCATGCATGAAGCGCTTCATAACCATTACACACAAAAATCTCTCTCACTGTCTCCCG GAAAGAAGGACCCCSEQ ID NO: 35, IgG1 CH₂CH₃ aaDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPSEQ ID NO: 36, Linker sequence (between scFv and CH₂CH₃) nt CTCGAGSEQ ID NO: 37, Linker sequence (between scFv and CH₂CH₃) aa LESEQ ID NO: 38, CD3 ζ transmembrane and cytoplasmicregions nt (transmembrane region is indicated by underline).AAACTGTGTTACCTCCTCGATGGCATCCTCTTTATTTATGGCGTGATTCTGACCGCATTGTTTCTCCGAGTAAAATTCTCTAGATCCGCAGACGCTCCCGCATATCAGCAAGGACAAAATCAGCTTTATAACGAACTTAACCTCGGCAGACGCGAAGAATACGATGTACTGGACAAGAGAAGAGGAAGAGATCCCGAAATGGGCGGAAAACCCCAGAGAAGAAAGAATCCCCAAGAAGGTCTTTATAACGAACTGCAGAAAGATAAAATGGCCGAAGCGTACAGTGAAATTGGTATGAAAGGAGAAAGAAGACGCGGAAAAGGACATGACGGACTCTACCAAGGACTCTCAACTGCTACTAAAGATACATACGACGCCCTTCATATGCAAGCCCTCCCCC CGAGATAASEQ ID NO: 39, CD3 ζ transmembrane and cytoplasmicregions aa (transmembrane region is indicated by underline).KLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Additional Chimeric Stimulating Molecule Sequences

SEQ ID NO: 40, OX40 ntGTTGCCGCCATCCTGGGCCTGGGCCTGGTGCTGGGGCTGCTGGGCCCCCTGGCCATCCTGCTGGCCCTGTACCTGCTCCGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATC SEQ ID NO: 41, OX40 aaVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQ EEQADAHSTLAKISEQ ID NO: 42, SEQ ID NO: 22 nucleotide sequence of 5′LTR sequenceTGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAAAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTATGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA

Additional Sequences

SEQ ID NO, 43 Thosea asigna virus-2A from capsid protein precursor nucleotide sequenceGCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCSEQ ID NO: 44, Thosea asigna virus-2A from capsid protein precursor amino acid sequenceAEGRGSLLTCGDVEENPGP SEQ ID NO: 45, 3′LTR nucleotide sequenceTGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCAGGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCASEQ ID NO: 46, (nucleotide sequence of linker-F_(v)1-F_(v)2-linker with XhoI/SalI sites, (wobbledcodons lowercase in F_(v)2′))CTCGAGTCTGGCGGTGGATCCGGAGGCGTTCAAGTAGAAACAATCAGCCCAGGAGACGGAAGGACTTTCCCCAAACGAGGCCAAACATGCGTAGTTCATTATACTGGGATGCTCGAAGATGGAAAAAAAGTAGATAGTAGTAGAGACCGAAACAAACCATTTAAATTTATGTTGGGAAAACAAGAAGTAATAAGGGGCTGGGAAGAAGGTGTAGCACAAATGTCTGTTGGCCAGCGCGCAAAACTCACAATTTCTCCTGATTATGCTTACGGAGCTACCGGCCACCCCGGCATCATACCCCCTCATGCCACACTGGTGTTTGACGTCGAATTGCTCAAACTGGAAGTCGAGGGaGTgCAgGTgGAgACgATtAGtCCtGGgGAtGGgAGaACcTTtCCaAAgCGcGGtCAgACcTGtGTtGTcCAcTAcACcGGtATGCTgGAgGAcGGgAAgAAgGTgGActcTtcacGcGAtCGcAAtAAgCCtTTcAAgTTcATGcTcGGcAAgCAgGAgGTgATccGGGGgTGGGAgGAgGGcGTgGCtCAgATGTCgGTcGGgCAaCGaGCgAAgCTtACcATcTCaCCcGAcTAcGCgTAtGGgGCaACgGGgCAtCCgGGaATtATcCCtCCcCAcGCtACgCTcGTaTTcGAtGTgGAgcTcttgAAgCTtGagTCTGGCGGTGGATCCGGAGTCGACSEQ ID NO: 47, (F_(V′)F_(VLS) amino acid sequence)LESGGGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEVEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDSEQ ID NO: 48, FKBPv36 (Fv1) nucleotide sequenceGGCGTTCAAGTAGAAACAATCAGCCCAGGAGACGGAAGGACTTTCCCCAAACGAGGCCAAACATGCGTAGTTCATTATACTGGGATGCTCGAAGATGGAAAAAAAGTAGATAGTAGTAGAGACCGAAACAAACCATTTAAATTTATGTTGGGAAAACAAGAAGTAATAAGGGGCTGGGAAGAAGGTGTAGCACAAATGTCTGTTGGCCAGCGCGCAAAACTCACAATTTCTCCTGATTATGCTTACGGAGCTACCGGCCACCCCGGCATCATACCCCCTCATGCCACACTGGTGTTTGACGTCGAATTGCTCA AACTGGAASEQ ID NO: 49, FKBPv36 (Fv1) amino acid sequenceGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESEQ ID NO: 50, FKBPv36 (Fv2) nucleotide sequenceGGaGTgCAgGTgGAgACgATtAGtCCtGGgGAtGGgAGaACcTTtCCaAAgCGcGGtCAgACcTGtGTtGTcCAcTAcACcGGtATGCTgGAgGAcGGgAAgAAgGTgGActcTtcacGcGAtCGcAAtAAgCCtTTcAAgTTcATGcTcGGcAAgCAgGAgGTgATccGGGGgTGGGAgGAgGGcGTgGCtCAgATGTCgGTcGGgCAaCGaGCgAAgCTtACcATcTCaCCcGAcTAcGCgTAtGGgGCaACgGGgCAtCCgGGaATtATcCCtCCcCAcGCtACgCTcGTaTTcGAtGTgGAgcTcttgAAgCTtGagSEQ ID NO: 51, FKBPv36 (Fv2) amino acid sequenceGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESEQ ID NO: 52, Myristoylation polypeptide nucleotide sequenceATGGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAGCGCSEQ ID NO: 53, Myristoylation polypeptide amino acid sequenceMGSSKSKPKDPSQR SEQ ID NO: 54, Linker nucleotide sequence (linker 1)CTCGAG SEQ ID NO: 55, Linker amino acid sequence (linker 1) LESEQ ID NO: 56, Truncated MyD88 polypeptide nucleotide sequenceATGGCCGCTGGGGGCCCAGGCGCCGGATCAGCTGCTCCCGTATCTTCTACTTCTTCTTTGCCGCTGGCTGCTCTGAACATGCGCGTGAGAAGACGCCTCTCCCTGTTCCTTAACGTTCGCACACAAGTCGCTGCCGATTGGACCGCCCTTGCCGAAGAAATGGACTTTGAATACCTGGAAATTAGACAACTTGAAACACAGGCCGACCCCACTGGCAGACTCCTGGACGCATGGCAGGGAAGACCTGGTGCAAGCGTTGGACGGCTCCTGGATCTCCTGACAAAACTGGGACGCGACGACGTACTGCTTGAACTCGGACCTAGCATTGAAGAAGACTGCCAAAAATATATCCTGAAACAACAACAAGAAGAAGCCGAAAAACCTCTCCAAGTCGCAGCAGTGGACTCATCAGTACCCCGAACAGCTGAGCTTGCTGGGATTACTACACTCGACGACCCACTCGGACATATGCCTGAAAGATTCGACGCTTTCATTTGCTATTGCCCCTCTGACATASEQ ID NO: 57, Truncated MyD88 polypeptide amino acid sequenceMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDISEQ ID NO: 58, ΔCD40 polypeptide nucleotide sequenceAAGAAAGTTGCAAAGAAACCCACAAATAAAGCCCCACACCCTAAACAGGAACCCCAAGAAATCAATTTCCCAGATGATCTCCCTGGATCTAATACTGCCGCCCCGGTCCAAGAAACCCTGCATGGTTGCCAGCCTGTCACCCAAGAGGACGGAAAAGAATCACGGATTAGCGTACAAGAGAGACAASEQ ID NO: 59, ΔCD40 polypeptide amino acid sequenceKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSEQ ID NO: 60, Linker nucleotide sequence (linker 2)GTCGAGTCTGGCGGTGGATCCGGASEQ ID NO: 61, Linker amino acid sequence (linker 2) VESGGGSGSEQ ID NO: 62, FKBPv36 (Fv1) nucleotide sequenceGGCGTTCAAGTAGAAACAATCAGCCCAGGAGACGGAAGGACTTTCCCCAAACGAGGCCAAACATGCGTAGTTCATTATACTGGGATGCTCGAAGATGGAAAAAAAGTAGATAGTAGTAGAGACCGAAACAAACCATTTAAATTTATGTTGGGAAAACAAGAAGTAATAAGGGGCTGGGAAGAAGGTGTAGCACAAATGTCTGTTGGCCAGCGCGCAAAACTCACAATTTCTCCTGATTATGCTTACGGAGCTACCGGCCACCCCGGCATCATACCCCCTCATGCCACACTGGTGTTTGACGTCGAATTGCTCA AACTGGAASEQ ID NO: 63, FKBPv36 (Fv1) amino acid sequenceGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESEQ ID NO: 64, Linker nucleotide sequence (linker 3) GTCGAGSEQ ID NO: 65, Linker amino acid sequence (linker 3) VESEQ ID NO: 66, FKBPv36 (Fv2) nucleotide sequenceGGaGTgCAgGTgGAgACgATtAGtCCtGGgGAtGGgAGaACcTTtCCaAAgCGcGGtCAgACcTGtGTtGTcCAcTAcACcGGtATGCTgGAgGAcGGgAAgAAgGTgGActcTtcacGcGAtCGcAAtAAgCCtTTcAAgTTcATGcTcGGcAAgCAgGAgGTgATccGGGGgTGGGAgGAgGGcGTgGCtCAgATGTCgGTcGGgCAaCGaGCgAAgCTtACcATcTCaCCcGAcTAcGCgTAtGGgGCaACgGGgCAtCCgGGaATtATcCCtCCcCAcGCtACgCTcGTaTTcGAtGTgGAgcTcttgAAgCTtGagSEQ ID NO: 67, FKBPv36 (Fv2) amino acid sequenceGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESEQ ID NO: 68, Linker nucleotide sequence (linker 4)TCTGGCGGTGGATCCGGAGTCGACSEQ ID NO: 69, Linker amino acid sequence (linker 4) SGGGSGVDSEQ ID NO: 70, Furin protease consensus cleavage site nucleotide sequenceCGCGCAAAGCGTSEQ ID NO: 71, Furin protease consensus cleavage site amino acid sequenceRAKR SEQ ID NO: 72, V5 epitope nucleotide sequenceGGAAAACCTATACCTAATCCATTGCTGGGCTTAGACTCAACASEQ ID NO: 73, V5 epitope nucleotide sequence GKPIPNPLLGLDSTSEQ ID NO: 74, Linker nucleotide sequence (linker 5) GGCAGCGGAAGCSEQ ID NO: 75, Linker amino acid sequence (linker 5) GSGSSEQ ID NO: 76, P2A nucleotide sequenceGCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCTSEQ ID NO: 77, P2A amino acid sequence ATNFSLLKQAGDVEENPGPSEQ ID NO 78, Linker nucleotide sequence (linker 6) ACGCGTSEQ ID NO: 79, Linker amino acid sequence (linker 6) TRSEQ ID NO: 80, ΔCD19 nucleotide sequenceATGCCCCCTCCTAGACTGCTGTTTTTCCTGCTCTTTCTCACCCCAATGGAAGTTAGACCTGAGGAACCACTGGTCGTTAAAGTGGAAGAAGGTGATAATGCTGTCCTCCAATGCCTTAAAGGGACCAGCGACGGACCAACGCAGCAACTGACTTGGAGCCGGGAGTCCCCTCTCAAGCCGTTTCTCAAGCTGTCACTTGGCCTGCCAGGTCTTGGTATTCACATGCGCCCCCTTGCCATTTGGCTCTTCATATTCAATGTGTCTCAACAAATGGGTGGATTCTACCTTTGCCAGCCCGGCCCCCCTTCTGAGAAAGCTTGGCAGCCTGGATGGACCGTCAATGTTGAAGGCTCCGGTGAGCTGTTTAGATGGAATGTGAGCGACCTTGGCGGACTCGGTTGCGGACTGAAAAATAGGAGCTCTGAAGGACCCTCTTCTCCCTCCGGTAAGTTGATGTCACCTAAGCTGTACGTGTGGGCCAAGGACCGCCCCGAAATCTGGGAGGGCGAGCCTCCATGCCTGCCGCCTCGCGATTCACTGAACCAGTCTCTGTCCCAGGATCTCACTATGGCGCCCGGATCTACTCTTTGGCTGTCTTGCGGCGTTCCCCCAGATAGCGTGTCAAGAGGACCTCTGAGCTGGACCCACGTACACCCTAAGGGCCCTAAGAGCTTGTTGAGCCTGGAACTGAAGGACGACAGACCCGCACGCGATATGTGGGTAATGGAGACCGGCCTTCTGCTCCCTCGCGCTACCGCACAGGATGCAGGGAAATACTACTGTCATAGAGGGAATCTGACTATGAGCTTTCATCTCGAAATTACAGCACGGCCCGTTCTTTGGCATTGGCTCCTCCGGACTGGAGGCTGGAAGGTGTCTGCCGTAACACTCGCTTACTTGATTTTTTGCCTGTGTAGCCTGGTTGGGATCCTGCATCTTCAGCGAGCCCTTGTATTGCGCCGAAAAAGAAAACGAATGACTGACCCTACACGACGATT CTGASEQ ID NO: 81, ΔCD19 amino acid sequenceMPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRF*

Example 6: Materials and Methods

Methods herein discuss the use of an inducible MyD88/CD40 construct, butmay also be used for the non-inducible MyD88/CD40 chimeric stimulatingmolecules with appropriate modifications.

Cell Lines, Media and Reagents.

293T (HEK 293T/17) and Capan-1, Raji, and Daudi cell lines were obtainedfrom the American Type Culture Collection. Cell lines were maintained inDMEM (Invitrogen, Grand Island, N.Y.) supplemented with 10% fetal calfserum (FCS) and 2 mM glutamax (Invitrogen) at 37° C. and 5% CO₂. T cellsgenerated from peripheral blood mononuclear cells (PBMC) were culturedin 45% RPMI 1640, 45% Click's media (Invitrogen) supplemented with 10%fetal bovine serum (FBS), 2 mM glutamax (T cell media; TCM) and 100 U/mlIL-2 (Miltenyi Biotec), unless otherwise noted. Clinical grade AP1903was diluted in ethanol to a 100 mM working solution for in vitro assays,or 0.9% saline for animal studies.

Retroviral and Plasmid Constructs.

Inducible MyD88/CD40 (iMC) comprising the myristoylation targetingsequence (M) (20), the TLR adaptor molecule MyD88, the CD40 cytoplasmicregion, and 2 tandem ligand-binding FKBP12v36 domains (Fv′Fv) werecloned in-frame with 2A-ΔCD19 in the SFG retroviral backbone usingGibson assembly (New England Biolabs, Ipswich, Mass.) to generateSFG-M.MyD88/CD40.Fv′Fv-2A-ΔCD19. Similarly, a control vector wasgenerated that contained only the myristoylation sequence and tandemFKBP12v36 binding domains (SFG-M.Fv′Fv-2A-ΔCD19). Additional retroviralvectors were constructed using a synthetic DNA approach (Integrated DNATechnologies, San Diego, Calif.) to generate MyD88 or CD40 onlyconstructs, termed SFG-M.MyD88.Fv′.Fv-2A-ΔCD19 orSFG-M.CD40.Fv′.Fv-2A-ΔCD19, respectively. Two first generation CARsrecognizing CD19 or PSCA were synthesized. The CD19 CAR was designedusing the anti-CD19 single chin fragment variable (scFv), FMC63, whilePSCA recognition was achieved using the murine scFv bm2B3. Both CARsincluded the IgG1 CH2CH3 spacer region, the CD28 transmembrane domainand CD3ζ cytoplasmic domain (PSCA.ζ) as discussed in Anurathapan et al.,2013 (13). A second generation CAR was constructed by PCR amplificationthat contained the CD28 transmembrane and cytoplasmic domain(PSCA.28.ζ). To generate a PSCA CAR that contained MC, MyD88/CD40 wassynthesized and inserted 5′ to CD3ζ (FIG. 3a ). For co-culture assays,Capan-1 tumor cells were modified by piggyBac transposase with a plasmidto express GFP (pIRII-GFP-2A-puromycin) and stably selected with 1 μg/mlpuromycin.

Retroviral Supernatant.

Retroviral supernatants were produced by transient co-transfection of293T cells with the SFG vector plasmid, Peg-Pam-e plasmid containing thesequence for MoMLV gag-pol and the RD114 plasmid encoding the RD114envelope using GeneJuice (EMD Biosciences, Gibbstown, N.J.) transfectionreagent as recommended by the manufacturer as previously discussed (14)Supernatant containing the retrovirus was collected 48 and 72 hoursafter transfection.

Generation of Activated T Cells.

Using peripheral blood mononuclear cells (PBMC) obtained from the GulfCoast Blood Bank (Houston, Tex.) anti-CD3/anti-CD28-activated T cellswere generated essentially as previously provided (22). Briefly, 5×10⁶PBMC resuspended in TCM and stimulated on non-tissue culture-treated24-well plates coated with 0.5 μg/ml each of anti-CD3 and anti-CD28antibodies (Miltenyi Biotec) in the presence of 100 U/ml IL-2. On day 3,activated T cells were harvested and transduced with retrovirus vectorsor expanded in media supplemented with IL-2 as discussed below.

Transduction of T Cells.

Non-tissue culture treated 24 well plates were coated with 7 μg/mlRetronectin (Takara Bio, Otsu, Shiga, Japan) overnight at 4° C. Thewells were washed with phosphate-buffered saline then coated withretroviral supernatant. Subsequently, activated T cells were plated at3×10⁵ cells per well in viral supernatant supplemented with 100 U/mlIL-2. After three days in culture, cells were harvested and expanded intissue culture treated plates containing TCM plus 100 U/ml IL-2. For twoor three-gene transductions, the protocol is identical to above exceptthe wells were coated with equal amounts of each retroviral supernatantand activated T cells were then plated into each well containing equalamounts of viral supernatant supplemented with 100 U/ml IL-2.

Immunophenotyping.

Gene-modified T cells were analyzed for iMC transgene expression 10-14days post-transduction by using CD3-PerCP.Cy5 and CD19-PE (BioLegend).To detect CAR-transduced cells, T cells were also stained with anFc-specific monoclonal antibody conjugated to APC (JacksonImmunoResearch Laboratories, West Grove, Pa.), which recognizes the IgG1CH₂CH₃ component of the receptor. All flow cytometry was performed usingan LSRII flow cytometer (Becton Dickenson, East Rutherford, N.J.), andthe data analyzed using FlowJo (Tree star, Ashland, Oreg.).

Cytokine and Chemokine Production.

Production of IFN-γ, IL-2 and IL-6 by T cells modified with iMC, controlvectors or CAR-modified T cells were analyzed by ELISA per themanufacturer's protocol (eBioscience, San Diego, Calif.). In this assay,non-transduced T cells and iMC- or control vector-modified T cells wereactivated with 10 nM AP1903 or vehicle (EtOH), and supernatantscollected at 48 hours. For analysis of CAR-modified T cells, T cellswere cocultured with Capan-1 tumor cells at 1:1 T cell to tumor cellratios and supernatants were harvested at 24 and 48 hours.

Cytotoxicity Assay.

The specific cytotoxicity of CAR T cells against Capan-1 tumor cells wasmeasured in 6 hour and 24 hour lactate dehydrogenase (LDH) assays perthe manufacturer's recommendations (Clontech Laboratories, MountainView, Calif.) using effector to target (E:T) ratios ranging from 10:1 to0.5:1 and using Capan-1 as target cells.

Coculture Experiment.

To test the cytotoxicity, activation, proliferation and cytokineproduction following PSCA.ζ CAR activation, co-culture assays wereperformed co-culture assays with Capan-1-GFP tumor cells at E:T ratiosof 10:1, 5:1, 1:1, 1:5 and 1:10 in TCM in the absence of exogenous IL-2.After 7 days, all residual cells were collected by trypsinization,counted and stained with CD3 and Fc-specific antibodies and analyzed byflow cytometry. In addition, similar co-culture experiments wereperformed using CD19 CAR with and without MC costimulation. CAR-modifiedT cells were cultured for 7 days with CD19+ Raji or Daudi cell lines andsubsequently analyzed by flow cytometry for CD3⁺ and CD19⁺ cells.

Statistics.

Data are represented as mean±SEM. Data were analyzed using unpairedStudent's t test to calculate 2-tailed or 1-tailed P values to determinestatistical significance in differences when comparing 2 treatmentgroups in all assays. Data were analyzed using GraphPad Prism version5.0 software (GraphPad).

Example 7: Activation of Primary T Cells with Inducible MyD88, CD40, orMyD88/CD40

Methods herein discuss the use of an inducible MyD88/CD40 construct, andthe synergy obtrained when both MyD88 and CD40 polypeptides are combinedinto one chimeric stimulating molecule. These methods may also be usedfor the non-inducible MyD88/CD40 chimeric stimulating molecules withappropriate modifications.

In a parallel study, it was observed that a novel costimulatingmolecule, iMC, can provide controlled costimulation to T cells. Toexamine whether MyD88, CD40 or both molecules should be included asendodomains in potential CAR constructs, four distinct vectors weredesigned containing the AP1903-binding domains only (Fv′Fv), orgenetically fused with MyD88 (iMyD88), CD40 (iCD40) or with both MyD88and CD40 (iMC) (FIG. 3a ). CD3/CD28-activated T cells were transduced,and the transduction efficiency of each of the vectors was measured byflow cytometric detection of CD19 on the surface of CD3 T cells(CD3⁺CD19⁺), showing that each of the retroviruses were sufficientlyexpressed in primary T cells (57%-95%) compared to non-transduced Tcells (FIG. 3b ). The ability of iMyD88, iCD40 or iMC to activate Tcells following exposure to AP1903 by measuring IFN-γ and IL-6production by ELISA was then assayed. It was observed that onlyiMC-transduced T cells produced significant quantities of both IFN-γ andIL-6 following AP1903 activation, whereas neither NT, iMyD88, nor iCD40showed cytokine production (FIGS. 3c and d ). These data suggest thatMyD88 and CD40 synergize as activation signaling molecules in human Tcells, and that a CAR molecule should benefit from inclusion of thecomposite MC signaling domain.

Expression of MyD88/CD40 Chimeric Antigen Receptors and ChimericStimulating Molecules

The following examples discuss the compositions and methods relating toMyD88/CD40 chimeric antigen receptors and chimeric stimulatingmolecules, as provided in this application. Also included arecompositions and methods related to a Caspase-9-based safety switch, andits use in cells that express the MyD88/CD40 chimeric antigen receptorsor chimeric stimulating molecules.

Example 8: Design and Activity of MyD88/CD40 Chimeric Antigen ReceptorsDesign of MC-CAR Constructs

Based on the activation data from the inducible MyD88/CD40 experimentsdiscussed herein, the potential of MC signaling in a CAR molecule inplace of conventional endodomains (e.g., CD28 and 4-1BB) was examined.MC (without AP1903-binding FKBPv36 regions) was subcloned into thePSCA.ζ to emulate the position of the CD28 endodomain (FIG. 4a ).Retrovirus was generated for each of the three constructs, transducedhuman T cells and subsequently measured transduction efficiencydemonstrating that PSCA.MC.ζ could be expressed (FIGS. 4b and 4c ). Toconfirm that T cells bearing each of these CAR constructs retained theirability to recognize PSCA⁺ tumor cells, 6-hour cytotoxicity assays wereperformed, which showed lysis of Capan-1 target cells (FIG. 4d ).Therefore, the addition of MC into the cytoplasmic region of a CARmolecule does not affect CAR expression or the recognition of antigen ontarget cells.

MC costimulation enhances T cell killing, proliferation and survival inCAR-modified T cells As demonstrated in short-term cytotoxicity assays,each of the three CAR designs showed the capacity to recognize and lyseCapan-1 tumor cells. Cytolytic effector function in effector T cells ismediated by the release of pre-formed granzymes and perforin followingtumor recognition, and activation through CD3ζ is sufficient to inducethis process without the need for costimulation. First generation CAR Tcells (e.g., CARs constructed with only the CD3ζ cytoplasmic region) canlyse tumor cells; however, survival and proliferation is impaired due tolack of costimulation. Hence, the addition of CD28 or 4-1 BBco-stimulating domains constructs has significantly improved thesurvival and proliferative capacity of CAR T cells.

To examine whether MC can similarly provide costimulating signalsaffecting survival and proliferation, coculture assays were performedwith PSCA⁺ Capan-1 tumor cells under high tumor:T cell ratios (1:1, 1:5,1:10 T cell to tumor cell). When T cell and tumor cell numbers wereequal (1:1), there was efficient killing of Capan-1-GFP cells from allthree constructs compared to non-transduced control T cells (FIGS. 4aand b ). However, when the CAR T cells were challenged with high numbersof tumor cells (1:10), there was a significant reduction of Capan-1-GFPtumor cells only when the CAR molecule contained either MC or CD28 (FIG.4c ).

To further examine the mechanism of costimulation by these two CARs cellviability and proliferation was assayed. PSCA CARs containing MC or CD28showed improved survival compared to non-transduced T cells and the CD3ζonly CAR, and T cell proliferation by PSCA.MC.ζ and PSCA.28.ζ wassignificantly enhanced (FIGS. 4a and b ). As other groups have shownthat CARs that contain co-stimulating signaling regions produce IL-2, akey survival and growth molecule for T cells (4), ELISAs were performedon supernatants from CAR T cells challenged with Capan-1 tumor cells.Although PSCA.28.ζ produced high levels of IL-2, PSCA.MC.ζ signalingalso produced significant levels of IL-2, which likely contributes tothe observed T cell survival and expansion in these assays (FIG. 6c ).Additionally, IL-6 production by CAR-modified T cells was examined, asIL-6 has been implicated as a key cytokine in the potency and efficacyof CAR-modified T cells (15). In contrast to IL-2, PSCA.MC.ζ producedhigher levels of IL-6 compared to PSCA.28.ζ, consistent with theobservations that iMC activation in primary T cells induces IL-6 (FIG.6d .). Together, these data suggest that co-stimulation through MCproduces similar effects to that of CD28, whereby following tumor cellrecognition, CAR-modified T cells produce IL-2 and IL-6, which enhance Tcell survival

Immunotherapy using CAR-modified T cells holds great promise for thetreatment of a variety of malignancies. While CARs were first designedwith a single signaling domain (e.g., CD3ζ),(16-19) clinical trialsevaluating the feasibility of CAR immunotherapy showed limited clinicalbenefit.(1, 2, 20, 21) This has been primarily attributed to theincomplete activation of T cells following tumor recognition, whichleads to limited persistence and expansion in vivo.(22) To address thisdeficiency, CARs have been engineered to include another stimulatingdomain, often derived from the cytoplasmic portion of T cellcostimulating molecules including CD28, 4-1 BB, OX40, ICOS and DAP10,(4, 23-30) which allow CART cells to receive appropriate costimulationupon engagement of the target antigen. Indeed, clinical trials conductedwith anti-CD19 CARs bearing CD28 or 4-1BB signaling domains for thetreatment of refractory acute lymphoblastic leukemia (ALL) havedemonstrated impressive T cell persistence, expansion and serial tumorkilling following adoptive transfer. (6-8)

CD28 costimulation provides a clear clinical advantage for the treatmentof CD19⁺ lymphomas. Savoldo and colleagues conducted a CAR-T cellclinical trial comparing first (CD19.ζ) and second generation CARs(CD19.28.ζ) and found that CD28 enhanced T cell persistence andexpansion following adoptive transfer.31 One of the principal functionsof second generation CARs is the ability to produce IL-2 that supports Tcell survival and growth through activation of the NFAT transcriptionfactor by CD3ζ (signal 1), and NF-κB (signal 2) by CD28 or 4-1BB.32 Thissuggested other molecules that similarly activated NF-κB might be pairedwith the CD3ζ chain within a CAR molecule. Our approach has employed a Tcell costimulating molecule that was originally developed as an adjuvantfor a dendritic cell (DC) vaccine.(12, 33) For full activation orlicensing of DCs, TLR signaling is usually involved in the upregulationof the TNF family member, CD40, which interacts with CD40L onantigen-primed CD4⁺ T cells. Because iMC was a potent activator of NF-κBin DCs, transduction of T cells with CARs that incorporated MyD88 andCD40 might provide the required costimulation (signal 2) to T cells, andenhance their survival and proliferation.

A set of experiments was performed to examine whether MyD88, CD40 orboth components were required for optimum T cell stimulation using theiMC molecule. Remarkably, it was found that neither MyD88 nor CD40 couldsufficiently induce T cell activation, as measured by cytokineproduction (IL-2 and IL-6), but when combined as a single fusionprotein, could induce potent T cell activation (FIG. 3). A PSCA CARincorporating MC was constructed and subsequently compared its functionagainst a first (PSCA.ζ) and second generation (PSCA.28.ζ CAR. Here itwas found that MC enhanced survival and proliferation of CAR T cells toa comparable level as the CD28 endodomain, suggesting that costimulationwas sufficient (FIG. 4). While PSCA.MC.ζ CAR-transduced T cells producedlower levels of IL-2 than PSCA.28.ζ the secreted levels weresignificantly higher than non-transduced T cells and T cells transducedwith the PSCA. CAR (FIG. 6). On the other hand, PSCA.MC.ζ CAR-transducedT cells secreted significantly higher levels of IL-6, an importantcytokine associated with T cell activation, than PSCA.28.ζ transduced Tcells, indicating that MC conferred unique properties to CAR functionthat may translate to improved tumor cell killing in vivo. Whilemolecular analyses of the relevant signaling pathways still needs to beperformed, these experiments indicate that MC can activate NF-κB (signal2) following antigen recognition by the extracellular CAR domain.

FIG. 3. Design of inducible costimulating molecules and effect on T cellactivation. A) Four vectors were designed incorporating FKBPv36AP1903-binding domains (Fv′.Fv) alone, or with MyD88, CD40 or theMyD88/CD40 fusion protein. B) Transduction efficiency of primaryactivated T cells using CD3⁺CD19⁺ flow cytometric analysis. C) Analysisof IFN-γ production of modified T cells following activation with andwithout 10 nM AP1903. D) Analysis of IL-6 production of modified T cellsfollowing activation with and without 10 nM AP1903. * denotes a p valueof <0.05.

FIG. 4. Design and functional validation of MC-CAR. A) Three PSCA CARconstructs were designed incorporating only CD3ζ, or with CD28 or MCendodomains. B) Transduction efficiency (percentage) was measured byanti-CAR-APC (recognizing the IgG1 CH₂CH₃ domain). C) Flow cytometryanalysis demonstrating high transduction efficiency of T cells withPSCA.MC.ζ CAR. D) Analysis of specific lysis of PSCA⁺ Capan-1 tumorcells by CAR-modified T cells in a 6 hour LDH release assay at a ratioof 1:1 T cells to tumor cells. * denotes a p value of <0.05.

FIG. 4. MC-CAR modified T cells kill Capan-1 tumor cells in long-termcoculture assays. A) Flow cytometric analysis of CAR-modified andnon-transduced T cells cultured with Capan-1-GFP tumor cells after 7days in culture at a 1:1 ratio. Quantitation of viable GFP⁺ cells byflow cytometry in coculture assays at a 1:1 (B) and 1:10 (C) T cell totumor cell ratio. * denotes a p value of <0.05.

FIG. 6. MC and CD28 costimulation enhance T cell survival, proliferationand cytokine production. T cells isolated from 1:10 T cell to tumor cellcoculture assays were assayed for cell viability (A) and cell number (B)to assess survival and proliferation in response to tumor cell exposure.Supernatants from coculture assays were subsequently measured for IL-2(C) and IL-6 (D) production by ELISA. * denotes a p value of <0.05.

Apart from survival and growth advantages, MC-induced costimulation mayalso provide additional functions to CAR-modified T cells. Medzhitov andcolleagues recently demonstrated that MyD88 signaling was critical forboth Th1 and Th17 responses and that it acted via IL-1 to render CD4⁺ Tcells refractory to regulatory T cell (Treg)-driven inhibition.(34)Experiments with iMC show that IL-la and β are secreted following AP1903activation (data not shown). In addition, Martin et al demonstrated thatCD40 signaling in CD8⁺ T cells via Ras, PI3K and protein kinase C,result in NF-κB-dependent induction of cytotoxic mediators granzyme andperforin that lyse CD4⁺CD25⁺ Treg cells (35). Thus, MyD88 and CD40co-activation may render CAR-T cells resistant to the immunosuppressiveeffects of Treg cells, a function that could be critically important inthe treatment of solid tumors and other types of cancers.

In summary, MC can be incorporated into a CAR molecule and primary Tcells transduced with retrovirus can express PSCA.MC.ζ without overttoxicity or CAR stability issues. Further, MC appears to provide similarcostimulation to that of CD28, where transduced T cells show improvedsurvival, proliferation and tumor killing compared to T cells transducedwith a first generation CAR. Additional experiments to determine whetherMC adds additional benefits to CARs, such as resistance to theinhibitory effects of Treg cells may be considered.

FIG. 10 provides an example of a plasmid map for a MyD88/CD40 chimericantigen receptor.

Example 9: Chimeric Stimulating Molecules Co-Expressed in T Cells withCARS

A MyD88/CD40 chimeric stimulating molecule may also be expressed in acell along with a CAR, which may, for example, comprise the scFvpolypeptide, and the CD3-ζ chain. In this method, the CSM molecule isused in combination with a CAR, thereby segregating CAR signaling intotwo separate functions. This second function, provided by the CAR,provides antigen-specific cytotoxicity to the engineered T cells. Forexample, a CAR with specificity against PSMA may be expressed in T cellsalong with a MyD88/CD40 chimeric stimulating molecule. Also, theMyD88/CD40 CSM and the CAR portions may be transfected or transducedinto the cells either on the same vector, in cis, or on separatevectors, in trans. Thus, the two polypeptides may be expressed using twonucleic acids, such as, for example, two plasmids or two viruses, andthe T cells may be, for example, transfected twice, or in particularembodiments, the two nucleic acids may be co-transfected. In otherembodiments, the two polypeptides may be expressed in one nucleic acid,such as, for example, in the same plasmid or virus. The nucleic acid mayexpress the two polypeptides using two separate promoters, one for theCAR and one for the CSM. Or, in other embodiments, the two polypeptidesmay be expressed using the same promoter. In this embodiment, the twopolypeptides may be separated by a cleavable polypeptide, such as, forexample, a 2A sequence. The engineered T may, for example, beadministered to a subject to generate a specific immune response, forexample one directed against a prostate cancer tumor.

In some embodiments, the chimeric stimulating molecule does not compriseCD40. It is understood that the methods, constructs, polypeptide, andcells provided for the MyD88/CD40 chimeric stimulating molecules may bemodified as necessary for expression and use of the MyD88 chimericstimulating molecules.

MyD88/CD40 basal activity was found to be sufficient to stimulate a Tcell which expresses a CD19-binding chimeric antigen receptor. TheMyD88/CD40 vector used in the assay expressed a chimeric inducibleMyD88/CD40 polypeptide, and was designed to be inducible by AP1903. Inthe absence of AP1903, there was sufficient basal activity to provideco-stimulation to the CD19-CAR following encounter with tumor cells.

Example of a MyD88/CD40 costimulating polypeptide co-expressed on thesame vector as a chimeric antigen receptor

SFG-Myr.MC.2A.CD19scFv.CD34e.CD8stm.zeta sequenceSEQ ID NO: 82 Myristoylation atggggagtagcaagagcaagcctaaggaccccagccagcgcSEQ ID NO: 83 Myristoylation MGSSKSKPKDPSQR SEQ ID: 84 Linker ctcgacSEQ ID NO: 85 Linker LD SEQ ID NO: 86 Truncated MyD88 lacking the TIRdomain atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgatctgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgccttcatctgcta ttgccccagcgacatcSEQ ID NO: 87 Truncated MyD88 lacking the TIR domainMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 88 Linker gtcgag SEQ ID NO: 89 LinkerVE SEQ ID NO: 90 CD40 without extracellular regionaaaaaggtggccaagaagccaaccaataaggccccccaccccaagcaggagccccaggagatcaattttcccgacgatcttcctggctccaacactgctgctccagtgcaggagactttacatggatgccaaccggtcacccaggaggatggcaaagagagtcgcatctcagtgcaggagagacagSEQ ID NO: 91 CD40 without extracellular regionKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQED GKESRISVQERQSEQ ID NO: 92 Linker CCGCGG SEQ ID NO: 93 Linker PRSEQ ID NO: 94 T2A sequenceGAAGGCCGAGGGAGCCTGCTGACATGTGGCGATGTGGAGGAAAACCCAGG ACCASEQ ID NO: 95 T2A sequence EGRGSLLTCGDVEENPGPSEQ ID NO: 96 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGT CCAGTGTAGCAGGSEQ ID NO: 97 Signal peptide MEFGLSWLFLVAILKGVQCSRSEQ ID NO: 98 FMC63 variable light chainGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGG GGGACTAAGTTGGAAATAACASEQ ID NO: 99 FMC63 variable light chainDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGG GTKLEITSEQ ID NO: 100 Flexible linker GGCGGAGGAAGCGGAGGTGGGGGCSEQ ID NO: 101 Flexible linker GGGSGGGGSEQ ID NO: 102 FMC63 variable heavy chainGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCAC CGTCTCCTCASEQ ID NO: 103: FMC83 variable heavy chainEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYY YGGSYAMDYWGQGTSVTVSSSEQ ID NO: 104 Linker GGATCC SEQ ID NO: 105 Linker GSSEQ ID NO: 106 CD34 minimal epitopeGAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTSEQ ID NO: 107 CD34 minimal epitope ELPTQGTFSNVSTNVSSEQ ID NO: 108 CD8 α stalk domainCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGCGAC SEQ ID NO: 109 CD8 α stalk domainPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSEQ ID NO: 110 CD8 α transmembrane domainATCTATATCTGGGCACCTCTCGCTGGCACCTGTGGAGTCCTTCTGCTCAGCCTGGTTATTACTCTGTACTGTAATCACCGGAATCGCCGCCGCGTTTGTA AGTGTCCCAGGSEQ ID NO: 111 CD8 α transmembrane domainIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR SEQ ID NO: 112 Linker GTCGACSEQ ID NO: 113 Linker VD SEQ ID NO: 114 CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO: 115 CD3 zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR

Example 10: References

The following references are cited in, or provide additional informationthat may be relevant.

-   1. Till B G, Jensen M C, Wang J, et al: CD20-specific adoptive    immunotherapy for lymphoma using a chimeric antigen receptor with    both CD28 and 4-1BB domains: pilot clinical trial results. Blood    119:3940-50, 2012.-   2. Pule M A, Savoldo B, Myers G D, et al: Virus-specific T cells    engineered to coexpress tumor-specific receptors: persistence and    antitumor activity in individuals with neuroblastoma. Nat Med    14:1264-70, 2008.-   3. Kershaw M H, Westwood J A, Parker L L, et al: A phase I study on    adoptive immunotherapy using gene-modified T cells for ovarian    cancer. Clin Cancer Res 12:6106-15, 2006.-   4. Carpenito C, Milone M C, Hassan R, et al: Control of large,    established tumor xenografts with genetically retargeted human T    cells containing CD28 and CD137 domains. Proc Natl Acad Sci USA    106:3360-5, 2009.-   5. Song D G, Ye Q, Poussin M, et al: CD27 costimulation augments the    survival and antitumor activity of redirected human T cells in vivo.    Blood 119:696-706, 2012.-   6. Kalos M, Levine B L, Porter D L, et al: T cells with chimeric    antigen receptors have potent antitumor effects and can establish    memory in patients with advanced leukemia. Sci Transl Med 3:95ra73,    2011.-   7. Porter D L, Levine B L, Kalos M, et al: Chimeric antigen    receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med    365:725-33, 2011.-   8. Brentjens R J, Davila M L, Riviere I, et al: CD19-targeted T    cells rapidly induce molecular remissions in adults with    chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med    5:177ra38, 2013.-   9. Pule M A, Straathof K C, Dotti G, et al: A chimeric T cell    antigen receptor that augments cytokine release and supports clonal    expansion of primary human T cells. Mol Ther 12:933-41, 2005.-   10. Finney H M, Akbar A N, Lawson A D: Activation of resting human    primary T cells with chimeric receptors: costimulation from CD28,    inducible costimulator, CD134, and CD137 in series with signals from    the TCR zeta chain. J Immunol 172:104-13, 2004.-   11. Guedan S, Chen X, Madar A, et al: ICOS-based chimeric antigen    receptors program bipolar TH17/TH1 cells. Blood, 2014.-   12. Narayanan P, Lapteva N, Seethammagari M, et al: A composite    MyD88/CD40 switch synergistically activates mouse and human    dendritic cells for enhanced antitumor efficacy. J Clin Invest    121:1524-34, 2011.-   13. Anurathapan U, Chan R C, Hindi H F, et al: Kinetics of tumor    destruction by chimeric antigen receptor-modified T cells. Mol Ther    22:623-33, 2014.-   14. Craddock J A, Lu A, Bear A, et al: Enhanced tumor trafficking of    GD2 chimeric antigen receptor T cells by expression of the chemokine    receptor CCR2b. J Immunother 33:780-8, 2010.-   15. Lee D W, Gardner R, Porter D L, et al: Current concepts in the    diagnosis and management of cytokine release syndrome. Blood    124:188-95, 2014.-   16. Becker M L, Near R, Mudgett-Hunter M, et al: Expression of a    hybrid immunoglobulin-T cell receptor protein in transgenic mice.    Cell 58:911-21, 1989.-   17. Goverman J, Gomez S M, Segesman K D, et al: Chimeric    immunoglobulin-T cell receptor proteins form functional receptors:    implications for T cell receptor complex formation and activation.    Cell 60:929-39, 1990.-   18. Gross G, Waks T, Eshhar Z: Expression of immunoglobulin-T-cell    receptor chimeric molecules as functional receptors with    antibody-type specificity. Proc Natl Acad Sci USA 86:10024-8, 1989.-   19. Kuwana Y, Asakura Y, Utsunomiya N, et al: Expression of chimeric    receptor composed of immunoglobulin-derived V regions and T-cell    receptor-derived C regions. Biochem Biophys Res Commun 149:960-8,    1987.-   20. Jensen M C, Popplewell L, Cooper L J, et al: Antitransgene    rejection responses contribute to attenuated persistence of    adoptively transferred CD20/CD19-specific chimeric antigen receptor    redirected T cells in humans. Biol Blood Marrow Transplant    16:1245-56, 2010.-   21. Park J R, Digiusto D L, Slovak M, et al: Adoptive transfer of    chimeric antigen receptor re-directed cytolytic T lymphocyte clones    in patients with neuroblastoma. Mol Ther 15:825-33, 2007.-   22. Ramos C A, Dotti G: Chimeric antigen receptor (CAR)-engineered    lymphocytes for cancer therapy. Expert Opin Biol Ther 11:855-73,    2011.-   23. Finney H M, Lawson A D, Bebbington C R, et al: Chimeric    receptors providing both primary and costimulatory signaling in T    cells from a single gene product. J Immunol 161:2791-7, 1998.-   24. Hombach A, Wieczarkowiecz A, Marquardt T, et al: Tumor-specific    T cell activation by recombinant immunoreceptors: CD3 zeta signaling    and CD28 costimulation are simultaneously required for efficient    IL-2 secretion and can be integrated into one combined CD28/CD3 zeta    signaling receptor molecule. J Immunol 167:6123-31, 2001.-   25. Maher J, Brentjens R J, Gunset G, et al: Human T-lymphocyte    cytotoxicity and proliferation directed by a single chimeric    TCRzeta/CD28 receptor. Nat Biotechnol 20:70-5, 2002.-   26. Imai C, Mihara K, Andreansky M, et al: Chimeric receptors with    4-1BB signaling capacity provoke potent cytotoxicity against acute    lymphoblastic leukemia. Leukemia 18:676-84, 2004.-   27. Wang J, Jensen M, Lin Y, et al: Optimizing adoptive polyclonal T    cell immunotherapy of lymphomas, using a chimeric T cell receptor    possessing CD28 and CD137 costimulatory domains. Hum Gene Ther    18:712-25, 2007.-   28. Zhao Y, Wang Q J, Yang S, et al: A herceptin-based chimeric    antigen receptor with modified signaling domains leads to enhanced    survival of transduced T lymphocytes and antitumor activity. J    Immunol 183:5563-74, 2009.-   29. Milone M C, Fish J D, Carpenito C, et al: Chimeric receptors    containing CD137 signal transduction domains mediate enhanced    survival of T cells and increased antileukemic efficacy in vivo. Mol    Ther 17:1453-64, 2009.-   30. Yvon E, Del Vecchio M, Savoldo B, et al: Immunotherapy of    metastatic melanoma using genetically engineered GD2-specific T    cells. Clin Cancer Res 15:5852-60, 2009.-   31. Savoldo B, Ramos C A, Liu E, et al: CD28 costimulation improves    expansion and persistence of chimeric antigen receptor-modified T    cells in lymphoma patients. J Clin Invest 121:1822-6, 2011.-   32. Kalinski P, Hilkens C M, Wierenga E A, et al: T-cell priming by    type-1 and type-2 polarized dendritic cells: the concept of a third    signal. Immunol Today 20:561-7, 1999.-   33. Kemnade J O, Seethammagari M, Narayanan P, et al: Off-the-shelf    Adenoviral-mediated Immunotherapy via Bicistronic Expression of    Tumor Antigen and iMyD88/CD40 Adjuvant. Mol Ther, 2012.-   34. Schenten D, Nish S A, Yu S, et al: Signaling through the adaptor    molecule MyD88 in CD4⁺ T cells is required to overcome suppression    by regulatory T cells. Immunity 40:78-90, 2014.-   35. Martin S, Pahari S, Sudan R, et al: CD40 signaling in CD8⁺CD40⁺    T cells turns on contra-T regulatory cell functions. J Immunol    184:5510-8, 2010.

Example 11: Expression of MyD88/CD40 Costimulating Molecules in TCell-Receptor-Expressing Cells and Tumor Infiltrating Lymphocytes

The modified cells that express the MyD88/CD40 costimulating moleculesprovided herein may also express a T cell receptor. In these examples,the T cell receptor may be endogenous to the cell, or may be provided tothe cell through transfection or transformation with a nucleic acidcomprising a polynucleotide encoding a T cell receptor. In certainexamples, the T cell receptor may be expressed on the same nucleic acidvector as the MyD88/CD40 costimulating molecule. In further examples,the T cell receptor may be expressed on the same nucleic acid vector asa chimeric inducible Caspase-9 molecule. Methods provided herein forconstructing vectors which co-express, or separately express a chimericantigen receptor, MyD88/CD40 costimulating molecule, or inducibleCaspase-9 molecule, may be modified as appropriate for co-expression orseparate expression of the T cell receptor, MyD88/CD40 costimulatingmolecule, or inducible Caspase-9 molecule. In some examples, themodified cells are tumor infiltrating lymphocytes.

Example 12: Selective Apoptosis of the Modified Cells

The modified cells may be provided with a mechanism to remove some, orall of the cells if the patient experiences negative effects, and thereis a need to reduce, or stop treatment. These cells may be used for allCSM- or CAR-expressing modified T cells, or the cells may be providedwith this ability where the CAR is directed against antigens that havepreviously caused, or are at risk to cause, lethal on-target, off-organtoxicity, where there is a need for an option to rapidly terminatetherapy.

An example of a chimeric polypeptide that may be expressed in themodified cells is provided in FIG. 7. In this example, a singlepolypeptide is encoded by the nucleic acid vector. The inducibleCaspase-9 polypeptide is separated from the CAR polypeptide duringtranslation, due to skipping of a peptide bond. (Donnelly, M L 2001, J.Gen. Virol. 82:1013-25).

Vector Construction and Confirmation of Expression

A safety switch that can be stably and efficiently expressed in human Tcells is presented herein. Expression vectors suitable for use as atherapeutic agent were constructed that included a modified humanCaspase-9 activity fused to a human FK506 binding protein (FKBP), suchas, for example, FKBP12v36. The Caspase-9/FKBP12 hybrid activity can bedimerized using a small molecule pharmaceutical. Full length, truncated,and modified versions of the Caspase-9 activity were fused to the ligandbinding domain, multimerizing, dimerizing, dimerization, ormultimerization region, and inserted into the retroviral vectorMSCV.IRES.GRP, which also allows expression of the fluorescent marker,GFP.

The full-length inducible Caspase-9 molecule (F′-F—C-Casp9) includes 2,3, or more FK506 binding proteins (FKBPs—for example, FKBP12v36variants) linked with a Ser-Gly-Gly-Gly-Ser-Gly linker to the small andlarge subunit of the caspase molecule. Full-length inducible Caspase-9(F′F—C-Casp9.I.GFP) has a full-length Caspase-9, also includes a caspaserecruitment domain (CARD; GenBank NM001 229) linked to 2 12-kDa humanFK506 binding proteins (FKBP12; GenBank AH002 818) that contain an F36Vmutation. The amino acid sequence of one or more of the FKBPs (F′) wascodon-wobbled (e.g., the 3rd nucleotide of each amino acid codon wasaltered by a silent mutation that maintained the originally encodedamino acid) to prevent homologous recombination when expressed in aretrovirus. F′F—C-Casp9C3S includes a cysteine to serine mutation atposition 287 that disrupts its activation site. In constructs F′F-Casp9,F-C-Casp9, and F′-Casp9, either the caspase activation domain (CARD),one FKBP, or both, were deleted, respectively. All constructs werecloned into MSCV.IRES.GFP as EcoRI-XhoI fragments. Coexpression of theinducible Caspase-9 constructs of the expected size with the marker geneGFP in transfected 293T cells was demonstrated by Western blot using aCaspase-9 antibody specific for amino acid residues 299-318, presentboth in the full-length and truncated caspase molecules as well as aGFP-specific antibody.

An initial screen indicated that the full length iCasp9 could not bemaintained stably at high levels in T cells, possibly due to transducedcells being eliminated by the basal activity of the transgene. The CARDdomain is involved in physiologic dimerization of Caspase-9 molecules,by a cytochrome C and adenosine triphosphate (ATP)-driven interactionwith apoptotic protease-activating factor 1 (Apaf-1). Because of the useof a CID to induce dimerization and activation of the suicide switch,the function of the CARD domain is superfluous in this context andremoval of the CARD domain was investigated as a method of reducingbasal activity.

Using the iCasp9 Suicide Gene to Improve the Safety of Allodepleted TCells after Haploidentical Stem Cell Transplantation

Presented in this example are expression constructs and methods of usingthe expression constructs to improve the safety of allodepleted T cellsafter haploidentical stem cell transplantation. Similar methods may beused to express the Caspase-9 expression constructs in non allodepletedcells. A retroviral vector encoding iCasp9 and a selectable marker(truncated CD19) was generated as a safety switch for donor T cells.Even after allodepletion (using anti-CD25 immunotoxin), donor T cellscould be efficiently transduced, expanded, and subsequently enriched byCD19 immunomagnetic selection to >90% purity. The engineered cellsretained anti-viral specificity and functionality, and contained asubset with regulatory phenotype and function. Activating iCasp9 with asmall-molecule dimerizer rapidly produced >90% apoptosis. Althoughtransgene expression was downregulated in quiescent T cells, iCasp9remained an efficient suicide gene, as expression was rapidlyupregulated in activated (alloreactive) T cells.

Materials and Methods Generation of Allodepleted T Cells

Allodepleted cells were generated from healthy volunteers as previouslypresented. Briefly, peripheral blood mononuclear cells (PBMCs) fromhealthy donors were co-cultured with 30 Gy-irradiated recipient EpsteinBarr virus (EBV)-transformed lymphoblastoid cell lines (LCL) atresponder-to-stimulator ratio of 40:1 in serum-free medium (AIM V;Invitrogen, Carlsbad, Calif.). After 72 hours, activated T cells thatexpressed CD25 were depleted from the co-culture by overnight incubationin RFT5-SMPT-dgA immunotoxin. Allodepletion was considered adequate ifthe residual CD3⁺CD25⁺ population was <1% and residual proliferation by3H-thymidine incorporation was <10%.

Plasmid and Retrovirus

SFG.iCasp9.2A.CD19 consists of inducible Caspase-9 (iCasp9) linked, viaa cleavable 2A-like sequence, to truncated human CD19. iCasp9 consistsof a human FK506-binding protein (FKBP12; GenBank AH002 818) with anF36V mutation, connected via a Ser-Gly-Gly-Gly-Ser-Gly linker to humanCaspase-9 (CASP9; GenBank NM 001229). The F36V mutation increases thebinding affinity of FKBP12 to the synthetic homodimerizer, AP20187 orAP1903. The caspase recruitment domain (CARD) has been deleted from thehuman Caspase-9 sequence because its physiological function has beenreplaced by FKBP12, and its removal increases transgene expression andfunction. The 2A-like sequence encodes an 20 amino acid peptide fromThosea asigna insect virus, which mediates >99% cleavage between aglycine and terminal proline residue, resulting in 19 extra amino acidsin the C terminus of iCasp9, and one extra proline residue in the Nterminus of CD19. CD19 consists of full-length CD19 (GenBank NM 001770)truncated at amino acid 333 (TDPTRRF), which shortens theintracytoplasmic domain from 242 to 19 amino acids, and removes allconserved tyrosine residues that are potential sites forphosphorylation.

A stable PG13 clone producing Gibbon ape leukemia virus (Gal-V)pseudotyped retrovirus was made by transiently transfecting Phoenix Ecocell line (ATCC product # SD3444; ATCC, Manassas, Va.) withSFG.iCasp9.2A.CD19. This produced Eco-pseudotyped retrovirus. The PG13packaging cell line (ATCC) was transduced three times withEco-pseudotyped or retrovirus to generate a producer line that containedmultiple SFG.iCasp9.2A.CD19 proviral integrants per cell. Single cellcloning was performed, and the PG13 clone that produced the highesttiter was expanded and used for vector production.

Retroviral Transduction

Culture medium for T cell activation and expansion consisted of 45% RPMI1640 (Hyclone, Logan, Utah), 45% Clicks (Irvine Scientific, Santa Ana,Calif.) and 10% fetal bovine serum (FBS; Hyclone). Allodepleted cellswere activated by immobilized anti-CD3 (OKT3; Ortho Biotech,Bridgewater, N.J.) for 48 hours before transduction with retroviralvector Selective allodepletion was performed by co-culturing donor PBMCwith recipient EBV-LCL to activate alloreactive cells: activated cellsexpressed CD25 and were subsequently eliminated by anti-CD25immunotoxin. The allodepleted cells were activated by OKT3 andtransduced with the retroviral vector 48 hours later. Immunomagneticselection was performed on day 4 of transduction; the positive fractionwas expanded for a further 4 days and cryopreserved.

In small-scale experiments, non-tissue culture-treated 24-well plates(Becton Dickinson, San Jose, Calif.) were coated with OKT3 1 g/ml for 2to 4 hours at 37° C. Allodepleted cells were added at 1×10⁶ cells perwell. At 24 hours, 100 U/ml of recombinant human interleukin-2 (IL-2)(Proleukin; Chiron, Emeryville, Calif.) was added. Retroviraltransduction was performed 48 hours after activation. Non-tissueculture-treated 24-well plates were coated with 3.5 g/cm2 recombinantfibronectin fragment (CH-296; Retronectin; Takara Mirus Bio, Madison,Wis.) and the wells loaded twice with retroviral vector-containingsupernatant at 0.5 ml per well for 30 minutes at 37° C., following whichOKT3-activated cells were plated at 5×10⁵ cells per well in freshretroviral vector-containing supernatant and T cell culture medium at aratio of 3:1, supplemented with 100 U/ml IL-2. Cells were harvestedafter 2 to 3 days and expanded in the presence of 50 U/ml IL-2.

Scaling-Up Production of Gene-Modified Allodepleted Cells

Scale-up of the transduction process for clinical application usednon-tissue culture-treated T75 flasks (Nunc, Rochester, N.Y.), whichwere coated with 10 ml of OKT3 1 μg/ml or 10 ml of fibronectin 7 μg/mlat 4° C. overnight. Fluorinated ethylene propylene bags corona-treatedfor increased cell adherence (2PF-0072AC, American FluorosealCorporation, Gaithersburg, Md.) were also used. Allodepleted cells wereseeded in OKT3-coated flasks at 1×10⁶ cells/ml. 100 U/ml IL-2 was addedthe next day. For retroviral transduction, retronectin-coated flasks orbags were loaded once with 10 ml of retrovirus-containing supernatantfor 2 to 3 hours. OKT3-activated T cells were seeded at 1×10⁶ cells/mlin fresh retroviral vector-containing medium and T cell culture mediumat a ratio of 3:1, supplemented with 100 U/ml IL-2. Cells were harvestedthe following morning and expanded in tissue-culture treated T75 or T175flasks in culture medium supplemented with between about 50 to 100 U/mlIL-2 at a seeding density of between about 5×10⁵ cells/ml to 8×10⁵cells/ml.

CD19 Immunomagnetic Selection

Immunomagnetic selection for CD19 was performed 4 days aftertransduction. Cells were labeled with paramagnetic microbeads conjugatedto monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech,Auburn, Calif.) and selected on MS or LS columns in small scaleexperiments and on a CliniMacs Plus automated selection device in largescale experiments. CD19-selected cells were expanded for a further 4days and cryopreserved on day 8 post transduction. These cells werereferred to as “gene-modified allodepleted cells”.

Immunophenotyping and Pentamer Analysis

Flow cytometric analysis (FACSCalibur and CellQuest software; BectonDickinson) was performed using the following antibodies: CD3, CD4, CD8,CD19, CD25, CD27, CD28, CD45RA, CD45RO, CD56 and CD62L. CD19-PE (Clone4G7; Becton Dickinson) was found to give optimum staining and was usedin all subsequent analysis. A Non-transduced control was used to set thenegative gate for CD19. An HLA-pentamer, HLA-B8-RAKFKQLL (Proimmune,Springfield, Va.) was used to detect T cells recognizing an epitope fromEBV lytic antigen (BZLF1). HLA-A2-NLVPMVATV pentamer was used to detectT cells recognizing an epitope from CMV-pp65 antigen.

Induction of Apoptosis with Chemical Inducer of Dimerization, AP20187

Suicide gene functionality was assessed by adding a small moleculesynthetic homodimerizer, AP20187 (Ariad Pharmaceuticals; Cambridge,Mass.), at 10 nM final concentration the day following CD19immunomagnetic selection. AP1903 may also be used. Cells were stainedwith annexin V and 7-amino¬actinomycin (7-AAD)(BD Pharmingen) at 24hours and analyzed by flow cytometry. Cells negative for both annexin Vand 7-AAD were considered viable, cells that were annexin V positivewere apoptotic, and cells that were both annexin V and 7-AAD positivewere necrotic. The percentage killing induced by dimerization wascorrected for baseline viability as follows: Percentage killing=100%−(%Viability in AP20187-treated cells+% Viability in non-treated cells).

Assessment of Transgene Expression Following Extended Culture andReactivation

Cells were maintained in T cell medium containing 50 U/ml IL-2 until 22days after transduction. A portion of cells was reactivated on 24-wellplates coated with 1 g/ml OKT3 and 1 g/ml anti-CD28 (Clone CD28.2, BDPharmingen, San Jose, Calif.) for 48 to 72 hours. CD19 expression andsuicide gene function in both reactivated and non-reactivated cells weremeasured on day 24 or 25 post transduction.

In some experiments, cells also were cultured for 3 weeks posttransduction and stimulated with 30 {tilde over (G)}irradiatedallogeneic PBMC at a responder:stimulator ratio of 1:1. After 4 days ofco-culture, a portion of cells was treated with 10 nM AP20187. Killingwas measured by annexin V/7-AAD staining at 24 hours, and the effect ofdimerizer on bystander virus-specific T cells was assessed by pentameranalysis on AP20187-treated and untreated cells.

Optimal culture conditions for maintaining the immunological competenceof suicide gene-modified T cells must be determined and defined for eachcombination of safety switch, selectable marker and cell type, sincephenotype, repertoire and functionality can all be affected by thestimulation used for polyclonal T cell activation, the method forselection of transduced cells, and duration of culture.

Phase I Clinical Trial of Allodepleted T Cells Transduced with InducibleCaspase-9 Suicide Gene after Haploidentical Stem Cell Transplantation

This example presents results of a phase 1 clinical trial using analternative suicide gene strategy. Briefly, donor peripheral bloodmononuclear cells were co-cultured with recipient irradiatedEBV-transformed lymphoblastoid cells (40:1) for 72 hrs., allodepletedwith a CD25 immunotoxin and then transduced with a retroviralsupernatant carrying the iCasp9 suicide gene and a selection marker(ΔCD19); ΔCD19 allowed enrichment to >90% purity via immunomagneticselection.

An example of a protocol for generation of a cell therapy product isprovided herein.

Source Material

Up to 240 ml (in 2 collections) of peripheral blood was obtained fromthe transplant donor according to established protocols. In some cases,dependent on the size of donor and recipient, a leukopheresis wasperformed to isolate sufficient T cells. 10-30 cc of blood also wasdrawn from the recipient and was used to generate the Epstein Barr virus(EBV)-transformed lymphoblastoid cell line used as stimulator cells. Insome cases, dependent on the medical history and/or indication of a lowB cell count, the LCLs were generated using appropriate 1st degreerelative (e.g., parent, sibling, or offspring) peripheral bloodmononuclear cells.

Generation of Allodepleted Cells

Allodepleted cells were generated from the transplant donors aspresented herein. Peripheral blood mononuclear cells (PBMCs) fromhealthy donors were co-cultured with irradiated recipient Epstein Barrvirus (EBV)-transformed lymphoblastoid cell lines (LCL) atresponder-to-stimulator ratio of 40:1 in serum-free medium (AIM V;Invitrogen, Carlsbad, Calif.). After 72 hours, activated T cells thatexpress CD25 were depleted from the co-culture by overnight incubationin RFT5-SMPT-dgA immunotoxin. Allodepletion is considered adequate ifthe residual CD3⁺CD25⁺ population was <1% and residual proliferation by3H-thymidine incorporation was <10%.

Retroviral Production

A retroviral producer line clone was generated for the iCasp9-ΔCD19construct. A master cell-bank of the producer also was generated.Testing of the master-cell bank was performed to exclude generation ofreplication competent retrovirus and infection by Mycoplasma, HIV, HBV,HCV and the like. The producer line was grown to confluency, supernatantharvested, filtered, aliquoted and rapidly frozen and stored at −80° C.Additional testing was performed on all batches of retroviralsupernatant to exclude Replication Competent Retrovirus (RCR) and issuedwith a certificate of analysis, as per protocol.

Transduction of Allodepleted Cells

Allodepleted T-lymphocytes were transduced using Fibronectin. Plates orbags were coated with recombinant Fibronectin fragment CH-296(Retronectin™, Takara Shuzo, Otsu, Japan). Virus was attached toretronectin by incubating producer supernatant in coated plates or bags.Cells were then transferred to virus coated plates or bags. Aftertransduction allodepleted T cells were expanded, feeding them with IL-2twice a week to reach the sufficient number of cells as per protocol.

CD19 Immunomagnetic Selection

Immunomagnetic selection for CD19 was performed 4 days aftertransduction. Cells are labeled with paramagnetic microbeads conjugatedto monoclonal mouse anti-human CD19 antibodies (Miltenyi Biotech,Auburn, Calif.) and selected on a CliniMacs Plus automated selectiondevice. Depending upon the number of cells required for clinicalinfusion cells were either cryopreserved after the CliniMacs selectionor further expanded with IL-2 and cryopreserved on day 6 or day 8 posttransduction.

Freezing

Aliquots of cells were removed for testing of transduction efficiency,identity, phenotype and microbiological culture as required for finalrelease testing by the FDA. The cells were cryopreserved prior toadministration according to protocol.

Study Drugs RFT5-SMPT-dgA

RFT5-SMPT-dgA is a murine IgG1 anti-CD25 (IL-2 receptor a chain)conjugated via a hetero-bifunctional crosslinker[N-succinimidyloxycarbonyl-α-methyl-d-(2-pyridylthio) toluene] (SMPT) tochemically deglycosylated ricin A chain (dgA). RFT5-SMPT-dgA isformulated as a sterile solution at 0.5 mg/ml.

Synthetic Homodimerizer, AP1903

Mechanism of Action: AP1903-inducible cell death is achieved byexpressing a chimeric protein comprising the pro-domain-deleted portionof human (Caspase-9) protein receptor, which signals apoptotic celldeath, fused to a drug-binding domain derived from human FK506-bindingprotein (FKBP). This chimeric protein remains quiescent inside cellsuntil administration of AP1903, which cross-links the FKBP domains,initiating caspase signaling and apoptosis.

Toxicology: AP1903 has been evaluated as an Investigational New Drug(IND) by the FDA and has successfully completed a phase I clinicalsafety study. No significant adverse effects were noted when AP1903 wasadministered over a 0.01 mg/kg to 1.0 mglkg dose range.

Pharmacology/Pharmacokinetics: Patients received 0.4 mg/kg of AP1903 asa 2 h infusion—based on published PK data which show plasmaconcentrations of 10 ng/mL-I275 ng/mL over the 0.01 mg/kg to 1.0 mg/kgdose range with plasma levels falling to 18% and 7% of maximum at 0.5and 2 hrs post dose.

Side Effect Profile in Humans: No serious adverse events occurred duringthe Phase 1 study in volunteers. The incidence of adverse events wasvery low following each treatment, with all adverse events being mild inseverity. Only one adverse event was considered possibly related toAP1903. This was an episode of vasodilatation, presented as “facialflushing” for 1 volunteer at the 1.0 mg/kg AP1903 dosage. This eventoccurred at 3 minutes after the start of infusion and resolved after 32minutes duration. All other adverse events reported during the studywere considered by the investigator to be unrelated or to haveimprobable relationship to the study drug. These events included chestpain, flu syndrome, halitosis, headache, injection site pain,vasodilatation, increased cough, rhinitis, rash, gum hemorrhage, andecchymosis.

Patients developing grade 1 GvHD were treated with 0.4 mg/kg AP1903 as a2-hour infusion. Protocols for administration of AP1903 to patientsgrade 1 GvHD were established as follows. Patients developing GvHD afterinfusion of allodepleted T cells are biopsied to confirm the diagnosisand receive 0.4 mg/kg of AP1903 as a 2 h infusion. Patients with Grade IGvHD received no other therapy initially, however if they showedprogression of GvHD conventional GvHD therapy was administered as perinstitutional guidelines. Patients developing grades 2-4 GvHD wereadministered standard systemic immunosuppressive therapy perinstitutional guidelines, in addition to the AP1903 dimerizer drug.

Instructions for preparation and infusion: AP1903 for injection isobtained as a concentrated solution of 2.33 ml in a 3 ml vial, at aconcentration of 5 mg/mi, (i.e., 10.66 mg per vial). Prior toadministration, the calculated dose was diluted to 100 mL in 0.9% normalsaline for infusion. AP1903 for injection (0.4 mg/kg) in a volume of 100ml was administered via IV infusion over 2 hours, using a non-DEHP,non-ethylene oxide sterilized infusion set and infusion pump.

The iCasp9 suicide gene expression construct (e.g., SFG.iCasp9.2A.ΔCD19)consists of inducible Caspase-9 (iCasp9) linked, via a cleavable 2A-likesequence, to truncated human CD19 (ΔCD19). iCasp9 includes a humanFK506-binding protein (FKBP12; GenBank AH002 818) with an F36V mutation,connected via a Ser-Gly-Gly-Gly linker to human Caspase-9 (CASP9;GenBank NM 001229). The F36V mutation may increase the binding affinityof FKBP12 to the synthetic homodimerizer, AP20187 or API903. The caspaserecruitment domain (CARD) has been deleted from the human Caspase-9sequence and its physiological function has been replaced by FKBP12. Thereplacement of CARD with FKBP12 increases transgene expression andfunction. The 2A-like sequence encodes an 18 amino acid peptide fromThosea Asigna insect virus, which mediates >99% cleavage between aglycine and terminal proline residue, resulting in 17 extra amino acidsin the C terminus of iCasp9, and one extra proline residue in the Nterminus of CD19. ΔCD19 consists of full length CD19 (GenBank NM 001770)truncated at amino acid 333 (TDPTRRF), which shortens theintracytoplasmic domain from 242 to 19 amino acids, and removes allconserved tyrosine residues that are potential sites forphosphorylation.

In Vivo Studies

Three patients received iCasp9⁺ T cells after haplo-CD34⁺ stem celltransplantation (SCT), at dose levels between about 1×10⁶ to about 3×10⁶cells/kg.

Infused T cells were detected in vivo by flow cytometry (CD3⁺ ΔCD19⁺) orqPCR as early as day 7 after infusion, with a maximum fold expansion of170±5 (day 29±9 after infusion. Two patients developed grade I/II aGvHDand AP1903 administration caused >90% ablation of CD3⁺ ΔCD19⁺ cells,within 30 minutes of infusion, with a further log reduction within 24hours, and resolution of skin and liver aGvHD within 24 hrs, showingthat iCasp9 transgene was functional in vivo.

Ex vivo experiments confirmed this data. Furthermore, the residualallodepleted T cells were able to expand and were reactive to viruses(CMV) and fungi (Aspergillus fumigatus) (IFN-γ production). These invivo studies found that a single dose of dimerizer drug can reduce oreliminate the subpopulation of T cells causing GvHD, but can spare virusspecific CTLs, which can then re-expand.

Immune Reconstitution

Depending on availability of patient cells and reagents, immunereconstitution studies (Immunophenotyping, T and B cell function) may beobtained at serial intervals after transplant. Several parametersmeasuring immune reconstitution resulting from icaspase transducedallodepleted T cells will be analyzed. The analysis includes repeatedmeasurements of total lymphocyte counts, T and CD19 B cell numbers, andFACS analysis of T cell subsets (CD3, CD4, CD8, CD16, CD19, CD27, CD28,CD44, CD62L, CCR7, CD56, CD45RA, CD45RO, alpha/beta and gamma/delta Tcell receptors). Depending on the availability of a patients T cells Tregulatory cell markers such as CD41CD251FoxP3 also are analyzed.Approximately 10-60 ml of patient blood is taken, when possible, 4 hoursafter infusion, weekly for 1 month, monthly×9 months, and then at 1 and2 years. The amount of blood taken is dependent on the size of therecipient and does not exceed 1-2 cc/kg in total (allowing for bloodtaken for clinical care and study evaluation) at any one blood draw.

Modified Caspase-9 Polypeptides with Lower Basal Activity and MinimalLoss of Ligand IC50

Basal signaling, signaling in the absence of agonist or activatingagent, is prevalent in a multitude of biomolecules. For example, it hasbeen observed in more than 60 wild-type G protein coupled receptors(GPCRs) from multiple subfamilies [1], kinases, such as ERK and abl [2],surface immunoglobulins [3], and proteases. Basal signaling has beenhypothesized to contribute to a vast variety of biological events, frommaintenance of embryonic stem cell pluripotency, B cell development anddifferentiation [4-6], T cell differentiation [2, 7], thymocytedevelopment [8], endocytosis and drug tolerance [9], autoimmunity [10],to plant growth and development [11]. While its biological significanceis not always fully understood or apparent, defective basal signalingcan lead to serious consequences. Defective basal G_(s) proteinsignaling has led to diseases, such as retinitis pigmentosa, colorblindness, nephrogenic diabetes insipidus, familial ACTH resistance, andfamilial hypocalciuric hypercalcemia [12, 13].

Even though homo-dimerization of wild-type initiator Caspase-9 isenergetically unfavorable, making them mostly monomers in solution[14-16], the low-level inherent basal activity of unprocessed Caspase-9[15, 17] is enhanced in the presence of the Apaf-1-based “apoptosome”,its natural allosteric regulator [6]. Moreover, supra-physiologicalexpression levels and/or co-localization could lead to proximity-drivendimerization, further enhancing basal activation. The modified cells ofthe present application may comprise nucleic acids coding for a chimericCaspase-9 polypeptide having lower basal signaling activity. Examples ofCaspase-9 mutants with lower basal signaling are provided in the tablebelow. Polynucleotides comprising Caspase-9 mutants with lower basalsignaling may be expressed in the modified cells used for cell therapyherein. In these examples, the modified cells may include a safetyswitch, comprising a polynucleotide encoding a lower basal signalingchimeric Caspase-9 polypeptide. In the event of an adverse eventfollowing administration of the modified cells comprising the chimericstimulating molecules or chimeric antigen receptors herein, Caspase-9activity may be induced by administering the dimerizer to the patient,thus inducing apoptosis and clearance of some, or all of the modifiedcells. In some examples, the amount of dimerizer administered may bedetermined as an amount designed to remove the highest amount, at least80% or 90% of the modified cells. In other examples, the amount ofdimerizer administered may be determined as an amount designed to removeonly a portion of the modified cells, in order to alleviate negativesymptoms or conditions, while leaving a sufficient amount of therapeuticmodified cells in the patient, in order to continue therapy. Methods forusing chimeric Caspase-9 polypeptides to induce apoptosis are discussedin PCT Application Number PCT/US2011/037381 by Malcolm K. Brenner etal., titled Methods for Inducing Selective Apoptosis, filed May 20,2011, and in U.S. patent application Ser. No. 13/112,739 by Malcolm K.Brenner et al., titled Methods for Inducing Selective Apoptosis, filedMay 20, 2011, issued Jul. 28, 2015 as U.S. Pat. No. 9,089,520. Chimericcaspase polypeptides having modified basal activity are discussed in PCTApplication Serial Number PCT/US2014/022004 by David Spencer et al.,titled Modified Caspase Polypeptides and Uses Thereof, filed Mar. 7,2014, published Oct. 9, 2014 as WO2014/164348, and in U.S. patentapplication Ser. No. 13/792,135 by David Spencer et al., titled ModifiedCaspase Polypeptides and Uses Thereof, filed Mar. 7, 2014; and in U.S.patent application Ser. No. 14/640,553 by Spencer et al., filed Mar. 6,2015. Methods for inducing partial apoptosis of the therapeutic modifiedcells are discussed in PCT Application Number PCT/US14/040964 by KevinSlawin et al., titled Methods for Inducing Partial Apoptosis UsingCaspase Polypeptides, filed Jun. 4, 2014, published Dec. 11, 2014 asWO2014/197638, and in U.S. patent application Ser. No. 14/296,404 byKevin Slawin et al., titled Methods for Inducing Partial Apoptosis UsingCaspase Polypeptides, filed Jun. 4, 2014. These patent applications andpublications are all incorporated by reference herein in theirentireties.

FIG. 10 provides a plasmid map for a MyD88/CD40 chimeric antigenreceptor co-expressed with a Caspase-9 polypeptide. FIG. 11 provides aplasmid map of a chimeric antigen receptor co-expressed with a Caspase-9polypeptide.

TABLE 1 Caspase Mutant Classes and Basal Activity HomodimerizationCleavage sites & Double mutants, Basal Activity domain XIAP InteractionPhosphorylation Misc. Decreased S144A basal and S144D similar IC₅₀ T317SS196D Decreased N405Q D330A S183A D330A-N405Q basal but ⁴⁰²GCFNF⁴⁰⁶ISAQT(Casp-10) D330E S195A D330A-S144A higher IC₅₀ F404Y D330G S196AD330A-S144D F406A D330N D330A-S183A F406W D330S D330A-S196A F406Y D330VN405Q-S144A N405Qco L329E N405Q-S144D T317A N405Q-S196D N405Q-T317S*N405Q-S144Aco *N405Q-T317Sco Decreased F404T D315A Y153A basal butF404W A316G Y153F much higher N405F F319W S307A IC₅₀ F406T Similar basalC403A ³¹⁶ATPF³¹⁹AVPI and IC₅₀ (SMAC/Diablo) C403S T317C C403T P318AN405A F319A Increased N405T T317E D330A-N405T basal F326K D327G D327KD327R Q328K Q328R L329G L329K A331K Catalytically ⁴⁰²GCFNF⁴⁰⁶AAAAA C285Adead ⁴⁰²GCFNF⁴⁰⁶YCSTL (Casp-2) D315A-D330A ⁴⁰²GCFNF⁴⁰⁶CIVSM (Casp-3)D330A-Y153A ⁴⁰²GCFNF⁴⁰⁶QPTFT (Casp-8) D330A-Y153F G402A D330A-T317EG402I G402Q G402Y C403P F404A F404S F406L Total mutants 80 *predictedBold, Tested in T cells

LITERATURE REFERENCES CITED OR PROVIDING ADDITIONAL SUPPORT TO THEPRESENT EXAMPLE

-   1. Seifert, R. and K. Wenzel-Seifert, Constitutive activity of    G-protein-coupled receptors: cause of disease and common property of    wild-type receptors. Naunyn Schmiedebergs Arch Pharmacol, 2002.    366(5): p. 381-416.-   2. Roose, J. P., et al., T cell receptor-independent basal signaling    via Erk and Abl kinases suppresses RAG gene expression. PLoS    Biol, 2003. 1(2): p. E53.-   3. Tze, L. E., et al., Basal immunoglobulin signaling actively    maintains developmental stage in immature B cells. PLoS Biol, 2005.    3(3): p. e82.-   4. Schram, B. R., et al., B cell receptor basal signaling regulates    antigen-induced Ig light chain rearrangements. J Immunol, 2008.    180(7): p. 4728-41.-   5. Randall, K. L., et al., Dock8 mutations cripple B cell    immunological synapses, germinal centers and long-lived antibody    production. Nat Immunol, 2009. 10(12): p. 1283-91.-   6. Kouskoff, V., et al., B cell receptor expression level determines    the fate of developing B lymphocytes: receptor editing versus    selection. Proc Natl Acad Sci USA, 2000. 97(13): p. 7435-9.-   7. Hong, T., et al., A simple theoretical framework for    understanding heterogeneous differentiation of CD4⁺ T cells. BMC    Syst Biol, 2012. 6: p. 66.-   8. Rudd, M. L., A. Tua-Smith, and D. B. Straus, Lck SH3 domain    function is required for T-cell receptor signals regulating    thymocyte development. Mol Cell Biol, 2006. 26(21): p. 7892-900.-   9. Sorkin, A. and M. von Zastrow, Endocytosis and signalling:    intertwining molecular networks. Nat Rev Mol Cell Biol, 2009.    10(9): p. 609-22.-   10. Luning Prak, E. T., M. Monestier, and R. A. Eisenberg, B cell    receptor editing in tolerance and autoimmunity. Ann N Y Acad    Sci, 2011. 1217: p. 96-121.-   11. Boss, W. F., et al., Basal signaling regulates plant growth and    development. Plant Physiol, 2010. 154(2): p. 439-43.-   12. Tao, Y. X., Constitutive activation of G protein-coupled    receptors and diseases: insights into mechanisms of activation and    therapeutics. Pharmacol Ther, 2008. 120(2): p. 129-48.-   13. Spiegel, A. M., Defects in G protein-coupled signal transduction    in human disease. Annu Rev Physiol, 1996. 58: p. 143-70.-   14. Shiozaki, E. N., et al., Mechanism of XIAP-mediated inhibition    of Caspase-9. Mol Cell, 2003. 11(2): p. 519-27.-   15. Renatus, M., et al., Dimer formation drives the activation of    the cell death protease Caspase-9. Proc Natl Acad Sci USA, 2001.    98(25): p. 14250-5.-   16. Shi, Y., Mechanisms of Caspase activation and inhibition during    apoptosis. Mol Cell, 2002. 9(3): p. 459-70.-   17. Shiozaki, E. N., J. Chai, and Y. Shi, Oligomerization and    activation of Caspase-9, induced by Apaf-1 CARD. Proc Natl Acad Sci    USA, 2002. 99(7): p. 4197-202.-   18. Straathof, K. C., et al., An inducible Caspase-9 safety switch    for T-cell therapy. Blood, 2005. 105(11): p. 4247-54.-   19. MacCorkle, R. A., K. W. Freeman, and D. M. Spencer, Synthetic    activation of Caspases: artificial death switches. Proc Natl Acad    Sci USA, 1998. 95(7): p. 3655-60.-   20. Di Stasi, A., et al., Inducible apoptosis as a safety switch for    adoptive cell therapy. N Engl J Med, 2011. 365(18): p. 1673-83.-   21. Chang, W. C., et al., Modifying ligand-induced and constitutive    signaling of the human 5-HT4 receptor. PLoS One, 2007. 2(12): p.    e1317.-   22. Bloom, J. D. and F. H. Arnold, In the light of directed    evolution: pathways of adaptive protein evolution. Proc Natl Acad    Sci USA, 2009. 106 Suppl 1: p. 9995-10000.-   23. Boatright, K. M. and G. S. Salvesen, Mechanisms of Caspase    activation. Curr Opin Cell Biol, 2003. 15(6): p. 725-31.-   24. Boatright, K. M., et al., A unified model for apical Caspase    activation. Mol Cell, 2003. 11(2): p. 529-41.-   25. Chao, Y., et al., Engineering a dimeric Caspase-9: a    re-evaluation of the induced proximity model for Caspase activation.    PLoS Biol, 2005. 3(6): p. e183.-   26. Stennicke, H. R., et al., Caspase-9 can be activated without    proteolytic processing. J Biol Chem, 1999. 274(13): p. 8359-62.-   27. Brady, S. C., L. A. Allan, and P. R. Clarke, Regulation of    Caspase-9 through phosphorylation by protein kinase C zeta in    response to hyperosmotic stress. Mol Cell Biol, 2005. 25(23): p.    10543-55.-   28. Martin, M. C., et al., Protein kinase A regulates Caspase-9    activation by Apaf-1 downstream of cytochrome c. J Biol Chem, 2005.    280(15): p. 15449-55.-   29. Cardone, M. H., et al., Regulation of cell death protease    Caspase-9 by phosphorylation. Science, 1998. 282(5392): p. 1318-21.-   30. Raina, D., et al., c-Abl tyrosine kinase regulates Caspase-9    autocleavage in the apoptotic response to DNA damage. J Biol    Chem, 2005. 280(12): p. 11147-51.-   31. Papworth, C., Bauer, J. C., Braman, J. and Wright, D. A.,    Site-directed mutagenesis in one day with >80% efficiency.    Strategies, 1996. 9(3): p. 3-4.-   32. Spencer, D. M., et al., Functional analysis of Fas signaling in    vivo using synthetic inducers of dimerization. Curr Biol, 1996.    6(7): p. 839-47.-   33. Hsiao, E. C., et al., Constitutive Gs activation using a    single-construct tetracycline-inducible expression system in    embryonic stem cells and mice. Stem Cell Res Ther, 2011. 2(2): p.    11.-   34. Waldner, C., et al., Double conditional human embryonic kidney    cell line based on FLP and PhiC31 mediated transgene integration.    BMC Res Notes, 2011. 4: p. 420.

Example 13: MC Costimulation Enhances Function and Proliferation of CD19CARs

FIGS. 34 and 40 show the results of experiments similar to thosediscussed herein, using an antigen recognition moiety that recognizesthe CD19 antigen. It is understood that the vectors provided herein maybe modified to construct a MyD88/CD40 CAR construct that targets CD19⁺tumor cells, which also incorporates an inducible Caspase-9 safetyswitch.

To examine whether MC costimulation functioned in CARs targeting otherantigens, T cells were modified with either CD19.ζ or with CD19.MC.ζ.The cytotoxicity, activation and survival against CD19+ Burkitt'slymphoma cell lines (Raji and Daudi) of the modified cells were assayed.In coculture assays, T cells transduced with either CAR showed killingof CD19+ Raji cells at an effector to target ratio as low as 1:1 (FIGS.21a and b ). However, analysis of cytokine production from co-cultureassays showed that CD19.MC.ζ transduced T cells produced higher levelsof IL-2 and IL-6 compared to CD19.ζ (FIGS. 21c and d ), which isconsistent with the costimulatory effects observed with iMC and PSCACARs containing the MC signaling domain. Further, T cells transducedwith CD19.MC.ζ showed enhanced proliferation following activation byRaji tumor cells (FIG. 21e ). These data support earlier experimentsdemonstrating that MC signaling in CAR molecules improves T cellactivation, survival and proliferation following ligation to a targetantigen expressed on tumor cells.

pBP0526-SFG.iCasp9wt.2A.CD19scFv.CD34e.CD8stm.MC.zeta (FIG. 22)SEQ ID NO: 116 FKBPv36ATGCTGGAGGGAGTGCAGGTGGAGACTATTAGCCCCGGAGATGGCAGAACATTCCCCAAAAGAGGACAGACTTGCGTCGTGCATTATACTGGAATGCTGGAAGACGGCAAGAAGGTGGACAGCAGCCGGGACCGAAACAAGCCCTTCAAGTTCATGCTGGGGAAGCAGGAAGTGATCCGGGGCTGGGAGGAAGGAGTCGCACAGATGTCAGTGGGACAGAGGGCCAAACTGACTATTAGCCCAGACTACGCTTATGGAGCAACCGGCCACCCCGGGATCATTCCCCCTCATGCTACACTGGTCTTCGATGTGGAGCTGCTGAAGCTGGAA SEQ ID NO: 117 FKBPv36MLEGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE SEQ ID NO: 118 LinkerAGCGGAGGAGGATCCGGA SEQ ID NO: 119 Linker SGGGSG SEQ ID NO: 120 Caspase-9GTGGACGGGTTTGGAGATGTGGGAGCCCTGGAATCCCTGCGGGGCAATGCCGATCTGGCTTACATCCTGTCTATGGAGCCTTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAGAGCGGGCTGCGGACCAGAACAGGATCCAATATTGACTGTGAAAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAGGTGAAAGGCGATCTGACCGCTAAGAAAATGGTGCTGGCCCTGCTGGAACTGGCTCGGCAGGACCATGGGGCACTGGATTGCTGCGTGGTCGTGATCCTGAGTCACGGCTGCCAGGCTTCACATCTGCAGTTCCCTGGGGCAGTCTATGGAACTGACGGCTGTCCAGTCAGCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCTTGCCCAAGTCTGGGCGGGAAGCCCAAACTGTTCTTTATTCAGGCCTGTGGAGGCGAGCAGAAAGATCACGGCTTCGAAGTGGCTAGCACCTCCCCCGAGGACGAATCACCTGGAAGCAACCCTGAGCCAGATGCAACCCCCTTCCAGGAAGGCCTGAGGACATTTGACCAGCTGGATGCCATCTCAAGCCTGCCCACACCTTCTGACATTTTCGTCTCTTACAGTACTTTCCCTGGATTTGTGAGCTGGCGCGATCCAAAGTCAGGCAGCTGGTACGTGGAGACACTGGACGATATCTTTGAGCAGTGGGCCCATTCTGAAGACCTGCAGAGTCTGCTGCTGCGAGTGGCCAATGCTGTCTCTGTGAAGGGGATCTACAAACAGATGCCAGGATGCTTCAACTTTCTGAGAAAGAAACTGTTCTTTAAGACCTCCGCATCTAGGGCCSEQ ID NO: 121 Caspase-9VDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSVVYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRA SEQ ID NO: 122 Linker CCGCGG SEQ ID NO: 123Linker PR SEQ ID NO: 124 T2AGAAGGCCGAGGGAGCCTGCTGACATGTGGCGATGTGGAGGAAAACCCAGGACCA SEQ ID NO: 125T2A EGRGSLLTCGDVEENPGP SEQ ID NO: 126 Linker CCATGG SEQ ID NO: 127Linker PW SEQ ID NO: 128 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGSEQ ID NO: 129 Signal peptide MEFGLSWLFLVAILKGVQCSR SEQ ID NO: 130FMC63 variable light chain (anti-CD19)GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAA TAACASEQ ID NO: 131 FMC63 variable light chain (anti CD19)DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO: 132 Flexible linkerGGCGGAGGAAGCGGAGGTGGGGGC SEQ ID NO: 133 Flexible linker GGGSGGGGSEQ ID NO: 134 FMC63 variable heavy chain (anti-CD19)GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA SEQ ID NO: 135FMC63 variable heavy chain (anti CD19)EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO: 136Linker GGATCC SEQ ID NO: 137 Linker GS SEQ ID NO: 138CD34 minimal epitope GAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTSEQ ID NO: 139 CD34 minimal epitope ELPTQGTFSNVSTNVS SEQ ID NO: 140CD8 α stalk domainCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGC GACSEQ ID NO: 141 CD8 α stalk domainPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 142CD8 α transmembrane domainATCTATATCTGGGCACCTCTCGCTGGCACCTGTGGAGTCCTTCTGCTCAGCCTGGTTATTACTCTGTACTGTAATCACCGGAATCGCCGCCGCGTTTGTAAGTGTCCCAGG SEQ ID NO: 143CD8 α transmembrane domain IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRSEQ ID NO: 144 Linker GTCGAC SEQ ID NO: 145 Linker VD SEQ ID NO: 146Truncated MyD88 lacking the TIR domainATGGCCGCTGGGGGCCCAGGCGCCGGATCAGCTGCTCCCGTATCTTCTACTTCTTCTTTGCCGCTGGCTGCTCTGAACATGCGCGTGAGAAGACGCCTCTCCCTGTTCCTTAACGTTCGCACACAAGTCGCTGCCGATTGGACCGCCCTTGCCGAAGAAATGGACTTTGAATACCTGGAAATTAGACAACTTGAAACACAGGCCGACCCCACTGGCAGACTCCTGGACGCATGGCAGGGAAGACCTGGTGCAAGCGTTGGACGGCTCCTGGATCTCCTGACAAAACTGGGACGCGACGACGTACTGCTTGAACTCGGACCTAGCATTGAAGAAGACTGCCAAAAATATATCCTGAAACAACAACAAGAAGAAGCCGAAAAACCTCTCCAAGTCGCAGCAGTGGACTCATCAGTACCCCGAACAGCTGAGCTTGCTGGGATTACTACACTCGACGACCCACTCGGACATATGCCTGAAAGATTCGACGCTTTCATTTGCTATTGCCCCTCTGACATA SEQ ID NO: 147 Truncated MyD88 lacking the TIR domainMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 148CD40 without the extracellular domainAAGAAAGTTGCAAAGAAACCCACAAATAAAGCCCCACACCCTAAACAGGAACCCCAAGAAATCAATTTCCCAGATGATCTCCCTGGATCTAATACTGCCGCCCCGGTCCAAGAAACCCTGCATGGTTGCCAGCCTGTCACCCAAGAGGACGGAAAAGAATCACGGATTAGCGTACAAGAGAGACAASEQ ID NO: 149 CD40 without the extracellular domainKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSEQ ID NO: 150 CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO: 151 CD3 zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 152Casp 9 (truncated) nucleotide sequenceGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCA SEQ ID NO: 153Caspase-9 (truncated) amino acid sequence-CARD domain deletedG F G D V G A L E S L R G N A D L A Y I L S M E P C G H C L I I N N V NF C R E S G L R T R T G S N I D C E K L R R R F S S L H F M V E V K G DL T A K K M V L A L L E L A Q Q D H G A L D C C V V V I L S H G C Q A SH L Q F P G A V Y G T D G C P V S V E K I V N I F N G T S C P S L G G KP K L F F I Q A C G G E Q K D H G F E V A S T S P E D E S P G S N P E PD A T P F Q E G L R T F D Q L D A I S S L P T P S D I F V S Y S T F P G FV S W R D P K S G S W Y V E T L D D I F E Q W A H S E D L Q S L L L RV A N A V S V K G I Y K Q M P G C F N F L R K K L F F K T S

Example 14: Cytokine Production of T Cells Co-Expressing a MyD88/CD40Chimeric Antigen Receptor and Inducible Caspase-9 Polypeptide

Various chimeric antigen receptor constructs were created to comparecytokine production of transduced T cells after exposure to antigen. Thechimeric antigen receptor constructs all had an antigen recognitionregion that bound to CD19. FIG. 30 provides schematics of the variousretroviral constructs used in the assays. FIG. 12 compares transductionefficiency and chimeric antigen receptor expression. FIG. 13 comparesIL-2 and IL-6 secretion after the transduced T cells are exposed toDaudi Burkitt's lymphoma cells. FIG. 14 provides the results of anotherassay comparing IL-2 and IL-6 secretion after the transduced T cells areexposed to Raji Burkitt's lymphoma cells. FIG. 15 shows the eliminationof CD19+ Daudi and Raji tumor cells following a co-culture withCAR-modified T cells.

Example 15: An Example of a MyD88/CD40 CAR Construct for Targeting Her2⁺Tumor Cells

FIGS. 18 and 19 show the results of experiments similar to thosediscussed herein, using an antigen recognition moiety that recognizesthe Her2/Neu antigen. It is understood that the vectors provided hereinmay be modified to construct a MyD88/CD40 CAR construct that targetsHer2⁺ tumor cells, which also incorporates an inducible Caspase-9 safetyswitch.

SFG-Her2scFv.CD34e.CD8stm.MC.zeta sequence SEQ ID NO: 152 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGSEQ ID NO: 153 Signal peptide MEFGLSWLFLVAILKGVQCSRSEQ ID NO: 154 FRP5 variable light chain (anti-Her2)GACATCCAATTGACACAATCACACAAATTTCTCTCAACTTCTGTAGGAGACAGAGTGAGCATAACCTGCAAAGCATCCCAGGACGTGTACAATGCTGTGGCTTGGTACCAACAGAAGCCTGGACAATCCCCAAAATTGCTGATTTATTCTGCCTCTAGTAGGTACACTGGGGTACCTTCTCGGTTTACGGGCTCTGGGTCCGGACCAGATTTCACGTTCACAATCAGTTCCGTTCAAGCTGAAGACCTCGCTGTTTATTTTTGCCAGCAGCACTTCCGAACCCCTTTTACTTTTGGCTCAGGCACTAAGTTGGAAATCAAGGCTTTG SEQ ID NO: 155 FRP5 variable light chain (anti-Her2)DIQLTQSHKFLSTSVGDRVSITCKASQDVYNAVAWYQQKPGQSPKLLIYSASSRYTGVPSRFTGSGSGPDFTFTISSVQAEDLAVYFCQQHFRTPFTFGSGTKLEIKALSEQ ID NO: 156 Flexible linker GGCGGAGGAAGCGGAGGTGGGGGCSEQ ID NO: 157 Flexible linker GGGSGGGGSEQ ID NO: 158 FRP5 variable heavy chain (anti-Her2/Neu)GAAGTCCAATTGCAACAGTCAGGCCCCGAATTGAAAAAGCCCGGCGAAACAGTGAAGATATCTTGTAAAGCCTCCGGTTACCCTTTTACGAACTATGGAATGAACTGGGTCAAACAAGCCCCTGGACAGGGATTGAAGTGGATGGGATGGATCAATACATCAACAGGCGAGTCTACCTTCGCAGATGATTTCAAAGGTCGCTTTGACTTCTCACTGGAGACCAGTGCAAATACCGCCTACCTTCAGATTAACAATCTTAAAAGCGAGGATATGGCAACCTACTTTTGCGCAAGATGGGAAGTTTATCACGGGTACGTGCCATACTGGGGACAAGGAACGACAGTGACAGTTAGTAGCSEQ ID NO: 159 FRP5 variable heavy chain (anti-Her2/Neu)EVQLQQSGPELKKPGETVKISCKASGYPFTNYGMNWVKQAPGQGLKWMGWINTSTGESTFADDFKGRFDFSLETSANTAYLQINNLKSEDMATYFCARWEVYHGYVPYWGQGTTVTVSSSEQ ID NO: 160 Linker GGATCC SEQ ID NO: 161 Linker GSSEQ ID NO: 162 CD34 minimal epitopeGAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTSEQ ID NO: 163 CD34 minimal epitope ELPTQGTFSNVSTNVSSEQ ID NO: 164 CD8 alpha stalkCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGC GACSEQ ID NO: 165 CD8 alpha stalkPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSEQ ID NO: 166 CD8 alpha transmembrane regionATCTATATCTGGGCACCTCTCGCTGGCACCTGTGGAGTCCTTCTGCTCAGCCTGGTTATTACTCTGTACTGTAATCACCGGAATCGCCGCCGCGTTTGTAAGTGTCCCAGGSEQ ID NO: 167 CD8 alpha transmembrane regionIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR SEQ ID NO: 168 Linker CtcgagSEQ ID NO: 169 Linker LE SEQ ID NO: 170 Truncated MyD88ATGGCCGCTGGGGGCCCAGGCGCCGGATCAGCTGCTCCCGTATCTTCTACTTCTTCTTTGCCGCTGGCTGCTCTGAACATGCGCGTGAGAAGACGCCTCTCCCTGTTCCTTAACGTTCGCACACAAGTCGCTGCCGATTGGACCGCCCTTGCCGAAGAAATGGACTTTGAATACCTGGAAATTAGACAACTTGAAACACAGGCCGACCCCACTGGCAGACTCCTGGACGCATGGCAGGGAAGACCTGGTGCAAGCGTTGGACGGCTCCTGGATCTCCTGACAAAACTGGGACGCGACGACGTACTGCTTGAACTCGGACCTAGCATTGAAGAAGACTGCCAAAAATATATCCTGAAACAACAACAAGAAGAAGCCGAAAAACCTCTCCAAGTCGCAGCAGTGGACTCATCAGTACCCCGAACAGCTGAGCTTGCTGGGATTACTACACTCGACGACCCACTCGGACATATGCCTGAAAGATTCGACGCTTTCATTTGCTATTGCCCCTCTGACATA SEQ ID NO: 171 Truncated MyD88MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDISEQ ID NO: 172 CD40 cytoplasmic domainAAGAAAGTTGCAAAGAAACCCACAAATAAAGCCCCACACCCTAAACAGGAACCCCAAGAAATCAATTTCCCAGATGATCTCCCTGGATCTAATACTGCCGCCCCGGTCCAAGAAACCCTGCATGGTTGCCAGCCTGTCACCCAAGAGGACGGAAAAGAATCACGGATTAGCGTACAAGAGAGACAASEQ ID NO: 173 CD40 cytoplasmic domainKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSEQ ID NO: 174 Linker gcggccgcagtcgag SEQ ID NO: 175 Linker AAAVESEQ ID NO: 176 CD3 zeta cytoplasmic domainAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCSEQ ID NO: 177 CD3 zeta cytoplasmic domainRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRExample 16: Additional Sequences SEQ ID NO: 178, ΔCasp9 (res. 135-416)G F G D V G A L E S L R G N A D L A Y I L S M E P C G H C L I I N N V NF C R E S G L R T R T G S N I D C E K L R R R F S S L H F M V E V K G DL T A K K M V L A L L E L A R Q D H G A L D C C V V V I L S H G C Q A SH L Q F P G A V Y G T D G C P V S V E K I V N I F N G T S C P S L G G KP K L F F I Q A C G G E Q K D H G F E V A S T S P E D E S P G S N P E PD A T P F Q E G L R T F D Q L D A I S S L P T P S D I F V S Y S T F P G FV S W R D P K S G S W Y V E T L D D I F E Q W A H S E D L Q S L L L RV A N A V S V K G I Y K Q M P G C F N F L R K K L F F K T SSEQ ID NO: 179, ΔCasp9 (res. 135-416) D330A, nucleotide sequenceGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCgGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGCCGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCASEQ ID NO: 180, ΔCasp9 (res. 135-416) D330A, amino acid sequenceG F G D V G A L E S L R G N A D L A Y I L S M E P C G H C L I I N N V NF C R E S G L R T R T G S N I D C E K L R R R F S S L H F M V E V K G DL T A K K M V L A L L E L A R Q D H G A L D C C V V V I L S H G C Q A SH L Q F P G A V Y G T D G C P V S V E K I V N I F N G T S C P S L G G KP K L F F I Q A C G G E Q K D H G F E V A S T S P E D E S P G S N P E PD A T P F Q E G L R T F D Q L A A I S S L P T P S D I F V S Y S T F P G FV S W R D P K S G S W Y V E T L D D I F E Q W A H S E D L Q S L L L RV A N A V S V K G I Y K Q M P G C F N F L R K K L F F K T SSEQ ID NO: 181, ΔCasp9 (res. 135-416) N405Q nucleotide sequenceGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCgGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTCAGTTCCTCCGGAAAAAACTTTTCTTTAAAACATCASEQ ID NO: 182, ΔCasp9 (res. 135-416) N405Q amino acid sequenceG F G D V G A L E S L R G N A D L A Y I L S M E P C G H C L I I N N V NF C R E S G L R T R T G S N I D C E K L R R R F S S L H F M V E V K G DL T A K K M V L A L L E L A R Q D H G A L D C C V V V I L S H G C Q A SH L Q F P G A V Y G T D G C P V S V E K I V N I F N G T S C P S L G G KP K L F F I Q A C G G E Q K D H G F E V A S T S P E D E S P G S N P E PD A T P F Q E G L R T F D Q L D A I S S L P T P S D I F V S Y S T F P G FV S W R D P K S G S W Y V E T L D D I F E Q W A H S E D L Q S L L L RV A N A V S V K G I Y K Q M P G C F Q F L R K K L F F K T SSEQ ID NO: 183, ΔCasp9 (res. 135-416) D330A N405Q nucleotide sequenceGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCgGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGCCGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTCAGTTCCTCCGGAAAAAACTTTTCTTTAAAACATCASEQ ID NO: 184, ΔCasp9 (res. 135-416) D330A N405Q amino acid sequenceG F G D V G A L E S L R G N A D L A Y I L S M E P C G H C L I I N N V NF C R E S G L R T R T G S N I D C E K L R R R F S S L H F M V E V K G DL T A K K M V L A L L E L A R Q D H G A L D C C V V V I L S H G C Q A SH L Q F P G A V Y G T D G C P V S V E K I V N I F N G T S C P S L G G KP K L F F I Q A C G G E Q K D H G F E V A S T S P E D E S P G S N P E PD A T P F Q E G L R T F D Q L A A I S S L P T P S D I F V S Y S T F P G FV S W R D P K S G S W Y V E T L D D I F E Q W A H S E D L Q S L L L RV A N A V S V K G I Y K Q M P G C F Q F L R K K L F F K T SSEQ ID NO: 185, Caspase-9.co nucleotide sequenceGTGGACGGGTTTGGAGATGTGGGAGCCCTGGAATCCCTGCGGGGCAATGCCGATCTGGCTTACATCCTGTCTATGGAGCCTTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAGAGCGGGCTGCGGACCAGAACAGGATCCAATATTGACTGTGAAAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAGGTGAAAGGCGATCTGACCGCTAAGAAAATGGTGCTGGCCCTGCTGGAACTGGCTCGGCAGGACCATGGGGCACTGGATTGCTGCGTGGTCGTGATCCTGAGTCACGGCTGCCAGGCTTCACATCTGCAGTTCCCTGGGGCAGTCTATGGAACTGACGGCTGTCCAGTCAGCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCTTGCCCAAGTCTGGGCGGGAAGCCCAAACTGTTCTTTATTCAGGCCTGTGGAGGCGAGCAGAAAGATCACGGCTTCGAAGTGGCTAGCACCTCCCCCGAGGACGAATCACCTGGAAGCAACCCTGAGCCAGATGCAACCCCCTTCCAGGAAGGCCTGAGGACATTTGACCAGCTGGATGCCATCTCAAGCCTGCCCACACCTTCTGACATTTTCGTCTCTTACAGTACTTTCCCTGGATTTGTGAGCTGGCGCGATCCAAAGTCAGGCAGCTGGTACGTGGAGACACTGGACGATATCTTTGAGCAGTGGGCCCATTCTGAAGACCTGCAGAGTCTGCTGCTGCGAGTGGCCAATGCTGTCTCTGTGAAGGGGATCTACAAACAGATGCCAGGATGCTTCCAGTTTCTGAGAAAGAAACTGTTCTTTAAGACCTCCGCATCTAGGGCCSEQ ID NO: 186, Caspase-9.co amino acid sequenceVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFQFLRKKLFFKTSASRASEQ ID NO: 187: Caspase9 D330E nucleotide sequenceGTCGACGGATTTGGTGATGTCGGTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCATTATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTGCGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTTTGCTGGAGCTGGCGCGGCAGGACCACGGTGCTCTGGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCCAGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCTTCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAAAGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACCCCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGcCGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTGTGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGACGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTGAAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCAGCTAGCAGAGCCSEQ ID NO: 188: Caspase9 D330E amino acid sequenceVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLeAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRASequences for pBPO509 (represented in FIG. 23)pBP0509-SFG-PSCAscFv.CH2CH3.CD28tm.zeta.MyD88/CD40 sequenceSEQ ID NO: 189 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGSEQ ID NO: 190 Signal peptide MEFGLSWLFLVAILKGVQCSRSEQ ID NO: 191 bm2B3 variable light chainGACATCCAGCTGACACAAAGTCCCAGTAGCCTGTCAGCCAGTGTCGGCGATAGGGTGACAATTACATGCTCCGCAAGTAGTAGCGTCAGATTCATACACTGGTACCAGCAGAAGCCTGGGAAGGCCCCAAAGAGGCTTATCTACGATACCAGTAAACTCGCCTCTGGAGTTCCTAGCCGGTTTTCTGGATCTGGCAGCGGAACTAGCTACACCCTCACAATCTCCAGTCTGCAACCAGAGGACTTTGCAACCTACTACTGCCAGCAATGGAGCAGCTCCCCTTTCACCTTTGGGCAGGGTACTAAGGTGGA GATCAAGSEQ ID NO: 192 bm2B3 variable light chainDIQLTQSPSSLSASVGDRVTITCSASSSVRFIHWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTSYTLTISSLQPEDFATYYCQQWSSSPFTFGQGTKVEIK SEQ ID NO: 193 Flexible linkerGGCGGAGGAAGCGGAGGTGGGGGC SEQ ID NO: 194 Flexible linker GGGSGGGGSEQ ID NO: 195 bm2B3 variable heavy chainGAGGTGCAGCTTGTAGAGAGCGGGGGAGGCCTCGTACAGCCAGGGGGCTCTCTGCGCCTGTCATGTGCAGCTTCAGGATTCAATATAAAGGACTATTACATTCACTGGGTACGGCAAGCTCCCGGTAAGGGCCTGGAATGGATCGGTTGGATCGACCCTGAAAACGGAGATACAGAATTTGTGCCCAAGTTCCAGGGAAAGGCTACCATGTCTGCCGATACTTCTAAGAATACAGCATACCTTCAGATGAATTCTCTCCGCGCCGAGGACACAGCCGTGTATTATTGTAAAACGGGAGGGTTCTGGGGTCAGGGTACCCTTGTGACTGTGTCTTCC SEQ ID NO: 196 bm2B3 variable heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFNIKDYYIHWVRQAPGKGLEWIGWIDPENGDTEFVPKFQGKATMSADTSKNTAYLQMNSLRAEDTAVYYCKTGGFWGQGTLVTVSS SEQ ID NO: 197 LinkerGGGGATCCCGCC SEQ ID NO: 198 Linker GDPA SEQ ID NO: 199 IgG1 hinge regionGAGCCCAAATCTCCTGACAAAACTCACACATGCCCA SEQ ID NO: 200 IgG1 hinge regionEPKSPDKTHTCP SEQ ID NO: 201 IgG1 CH2 regionCCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAAGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAATGCAAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA SEQ ID NO: 202 IgG1 CH2 regionPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKSEQ ID NO: 203 IgG1 CH3 regionGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 204 IgG1 CH3 regionGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 205 LinkerAAAGATCCCAAA SEQ ID NO: 206 Linker KDPKSEQ ID NO: 207 CD28 transmembrane regionTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT SEQ ID NO: 208 CD28 transmembrane regionFWVLVVVGGVLACYSLLVTVAFII SEQ ID NO: 209 Linker gccggcSEQ ID NO: 210 Linker AG SEQ ID NO: 211 CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO: 212 CD3 zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 213 MyD88GCCGCTGGGGGCCCAGGCGCCGGATCAGCTGCTCCCGTATCTTCTACTTCTTCTTTGCCGCTGGCTGCTCTGAACATGCGCGTGAGAAGACGCCTCTCCCTGTTCCTTAACGTTCGCACACAAGTCGCTGCCGATTGGACCGCCCTTGCCGAAGAAATGGACTTTGAATACCTGGAAATTAGACAACTTGAAACACAGGCCGACCCCACTGGCAGACTCCTGGACGCATGGCAGGGAAGACCTGGTGCAAGCGTTGGACGGCTCCTGGATCTCCTGACAAAACTGGGACGCGACGACGTACTGCTTGAACTCGGACCTAGCATTGAAGAAGACTGCCAAAAATATATCCTGAAACAACAACAAGAAGAAGCCGAAAAACCTCTCCAAGTCGCAGCAGTGGACTCATCAGTACCCCGAACAGCTGAGCTTGCTGGGATTACTACACTCGACGACCCACTCGGACATATGCCTGAAAGATTCGACGCTTTCATTTGCTATTGCCCCTCTGACATA SEQ ID NO: 214 213 MyD88AAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 215 CD40AAGAAAGTTGCAAAGAAACCCACAAATAAAGCCCCACACCCTAAACAGGAACCCCAAGAAATCAATTTCCCAGATGATCTCCCTGGATCTAATACTGCCGCCCCGGTCCAAGAAACCCTGCATGGTTGCCAGCCTGTCACCCAAGAGGACGGAAAAGAATCACGGATTAGCGTACAAGAGAGACAATA GSEQ ID NO: 216 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ*Sequences for pBPO425 (represented in FIG. 24)pBP0521-SFG-CD19scFv.CH2CH3.CD28tm.MyD88/CD40.zeta sequenceSEQ ID NO: 217 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGSEQID NO: 218 Signal peptide MEFGLSWLFLVAILKGVQCSRSEQ ID NO: 219 FMC63 variable light chain GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACASEQ ID NO: 220 FMC63 variable light chainDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO: 221 Flexible linkerGGCGGAGGAAGCGGAGGTGGGGGC SEQ ID NO: 222 Flexible linker GGGSGGGGSEQ ID NO: 223 FMC63 variable heavy chainGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCASEQ ID NO: 224 FMC63 variable heavy chainEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSSEQ ID NO: 225 Linker GGGGATCCCGCC SEQ ID NO: 226 Linker GDPASEQ ID NO: 227 IgG1 hinge GAGCCCAAATCTCCTGACAAAACTCACACATGCCCASEQ ID NO: 228 IgG1 hinge EPKSPDKTHTCP SEQ ID NO: 229 IgG1 CH2 regionCCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAAGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAATGCAAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAA SEQ ID NO: 230 IgG1 CH2 regionPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKSEQ ID NO: 231 IgG1 CH3 regionGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO: 232 IgG1 CH3 regionGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 233 LinkerAAAGATCCCAAA SEQ ID NO: 234 Linker KDPKSEQ ID NO: 235 CD28 transmembrane regionTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATT SEQ ID NO: 236 CD28 transmembrane regionFWVLVVVGGVLACYSLLVTVAFII SEQ ID NO: 237 Linker CtcgagSEQ ID NO: 238 Linker LE SEQ ID NO: 239 MyD88ATGGCCGCTGGGGGCCCAGGCGCCGGATCAGCTGCTCCCGTATCTTCTACTTCTTCTTTGCCGCTGGCTGCTCTGAACATGCGCGTGAGAAGACGCCTCTCCCTGTTCCTTAACGTTCGCACACAAGTCGCTGCCGATTGGACCGCCCTTGCCGAAGAAATGGACTTTGAATACCTGGAAATTAGACAACTTGAAACACAGGCCGACCCCACTGGCAGACTCCTGGACGCATGGCAGGGAAGACCTGGTGCAAGCGTTGGACGGCTCCTGGATCTCCTGACAAAACTGGGACGCGACGACGTACTGCTTGAACTCGGACCTAGCATTGAAGAAGACTGCCAAAAATATATCCTGAAACAACAACAAGAAGAAGCCGAAAAACCTCTCCAAGTCGCAGCAGTGGACTCATCAGTACCCCGAACAGCTGAGCTTGCTGGGATTACTACACTCGACGACCCACTCGGACATATGCCTGAAAGATTCGACGCTTTCATTTGCTATTGCCCCTCTGACATA SEQ ID NO: 240 MyD88MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 241 CD40AAGAAAGTTGCAAAGAAACCCACAAATAAAGCCCCACACCCTAAACAGGAACCCCAAGAAATCAATTTCCCAGATGATCTCCCTGGATCTAATACTGCCGCCCCGGTCCAAGAAACCCTGCATGGTTGCCAGCCTGTCACCCAAGAGGACGGAAAAGAATCACGGATTAGCGTACAAGAGAGACAASEQ ID NO: 242 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSEQ ID NO: 243 Linker gcggccgcagTCGAG SEQ ID NO: 244 Linker AAAVESEQ ID NO: 245 CD3 zeta chainAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA SEQ ID NO: 246 CD3 zeta chainRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*Sequences for SFG-Myr.MC-2A-CD19.scfv.CD34e.CD8stm.zeta (FIG. 25)SFG-Myr.MC.2A.CD19scFv.CD34e.CD8stm.zeta sequenceSEQ ID NO: 247 Myristolation atggggagtagcaagagcaagcctaaggaccccagccagcgcSEQ ID NO: 248 Myristolation MGSSKSKPKDPSQR SEQ ID NO: 249 Linker ctcgacSEQ ID NO: 250 Linker LD SEQ ID NO: 251 MyD88atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgatctgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgccttcatctgctattgccccagcgacatcSEQ ID NO:252 MyD88MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI SEQ ID NO: 253 Linker gtcgagSEQ ID NO: 254 Linker VE SEQ ID NO: 255 CD40aaaaaggtggccaagaagccaaccaataaggccccccaccccaagcaggagccccaggagatcaattttcccgacgatcttcctggctccaacactgctgctccagtgcaggagactttacatggatgccaaccggtcacccaggaggatggcaaagagagtcgcatctcagtgcaggagagacag SEQ ID NO: 256 CD40KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQSEQ ID NO: 257 Linker CCGCGG SEQ ID NO: 258 Linker PRSEQ ID NO: 259 T2A sequenceGAAGGCCGAGGGAGCCTGCTGACATGTGGCGATGTGGAGGAAAACCCAGGACCASEQ ID NO: 260 T2A sequence EGRGSLLTCGDVEENPGPSEQ ID NO: 261 Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGSEQ ID NO: 262 Signal peptide MEFGLSWLFLVAILKGVQCSRSEQ ID NO: 263 FMC63 variable light chainGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAA TAACASEQ ID NO: 264 FMC63 variable light chainDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT SEQ ID NO: 265 Flexible linkerGGCGGAGGAAGCGGAGGTGGGGGC SEQ ID NO: 266 Flexible linker GGGSGGGGSEQ ID NO: 267 FMC63 variable heavy chainGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCASEQ ID NO: 268 FMC63 variable heavy chainEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSSEQ ID NO: 269 Linker GGATCC SEQ ID NO: 270 Linker GSSEQ ID NO: 271 CD34 minimal epitopeGAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTSEQ ID NO: 272 CD34 minimal epitope ELPTQGTFSNVSTNVSSEQ ID NO: 273 CD8 alpha stalk domainCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGC GACSEQ ID NO: 274 CD8 alpha stalk domainPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSEQ ID NO: 275 CD8 alpha transmembrane domainATCTATATCTGGGCACCTCTCGCTGGCACCTGTGGAGTCCTTCTGCTCAGCCTGGTTATTACTCTGTACTGTAATCACCGGAATCGCCGCCGCGTTTGTAAGTGTCCCAGGSEQ ID NO: 276 CD8 alpha transmembrane domainIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR SEQ ID NO: 277 Linker GTCGACSEQ ID NO: 278 Linker VD SEQ ID NO: 279 CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC SEQ ID NO: 280 CD3 zetaRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID NO: 281 (MyD88 nucleotide sequence)atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgagctgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgccttcatctgctattgccccagcgacatccagtttgtgcaggagatgatccggcaactggaacagacaaactatcgactgaagttgtgtgtgtctgaccgcgatgtcctgcctggcacctgtgtctggtctattgctagtgagctcatcgaaaagaggtgccgccggatggtggtggttgtctctgatgattacctgcagagcaaggaatgtgacttccagaccaaatttgcactcagcctctctccaggtgcccatcagaagcgactgatccccatcaagtacaaggcaatgaagaaagagttccccagcatcctgaggttcatcactgtctgcgactacaccaacccctgcaccaaatcttggttctggactcgccttgccaaggccttgtccctgcccSEQ ID NO: 282 (MyD88 amino acid sequence)M A A G G P G A G S A A P V S S T S S L P L A A L N M R V R R R L S L F L N V R T Q V A A DW T A L A E E M D F E Y L E I R Q L E T Q A D P T G R L L D A W Q G R P G A S V G R L L E LL T K L G R D D V L L E L G P S I E E D C Q K Y I L K Q Q Q E E A E K P L Q V A A V D S S V PR T A E L A G I T T L D D P L G H M P E R F D A F I C Y C P S D I Q F V Q E M I R Q L E Q T NY R L K L C V S D R D V L P G T C V W S I A S E L I E K R C R R M V V V V S D D Y L Q S K EC D F Q T K F A L S L S P G A H Q K R L I P I K Y K A M K K E F P S I L R F I T V C D Y T N P CT K S W F W T R L A K A L S L P

Example 17: Modification of the Position of the MyD88/CD40 CostimulatoryDomain in CAR-Modified T Cells and Costimulatory Activity of aCytoplasmic Chimeric Stimulating Molecule

Examples presented herein evaluating the utility of MyD88/CD40 (MC)costimulation in CAR-T cells focused on including the MyD88/CD40polypeptide within the CAR, in the conventional location forcostimulatory domains such as CD28 or OX40, for example. In the presentexample, the polynucleotide encoding the MyD88/CD40 polypeptide wasplaced between the CD8 transmembrane region and CD3ζ (FIG. 26). This CARdesign (designated MC.ζ) demonstrated significantly higher cytokineproduction (e.g. IL-2 and IL-6), enhanced T cell survival andproliferation, and superior tumor killing during in vitro cocultureassays. However, the in vivo mouse studies showed that althoughantitumor activity was enhanced compared to CARs lacking costimulation,long term tumor control against CD19+ or Her2+ tumors was not achieved.

Following retroviral transduction, CAR expression (mean fluorescentintensity; MFI) is decreased with CARs containing the MyD88/CD40signaling domain. To assess whether basal activity from the MyD88/CD40domain or protein instability may be the cause of lower MFI intransduced T cells, T cells were transduced with vectors that encodedthe MyD88/CD40 signaling domain and CD3zeta (FIG. 27). Whether lower CARmolecule expression in transduced T cells adversely affects the functionand antitumor properties of the CAR, possibly contributing thesuboptimal tumor control in our animal studies, was also assessed.

In these experiments, a cytoplasmic chimeric stimulating molecule,MyD88/CD40, expressed constitutively, but separate from the CARmolecule, was tested to determine whether it could retain thecostimulatory properties while increasing CAR expression and stability.An additional vector was designed, SFG-iCasp9.2A.CAR.ζ.2A.MC thatproduces MyD88/CD40 constitutively using a P2A self-cleavage element.The MyD88/CD40 polypeptide is constitutively expressed, andconstitutively active, that is, it does not have a multimeric ligandbinding region and it stimulates immune activity without the need for aninducer. Construct designations in this example that include “MC” referto a polynucleotide sequence coding for a MyD88/CD40 polypeptide thatalso includes a myristoylation sequence—the functionality of thismyristoylation sequence, however, is destroyed due to the addition of aproline. (Resh, M. D., Biochim. Biophys. Acta. 1451: 1-16 (1999)).Initially, experiments evaluating the function of this design wasperformed with the CD19 scFv (FMC63), and then subsequently with a Her2scFv (FRPS). Following transduction, although all T cells wereefficiently transduced (>75%), T cells expressing the 2A form ofMyD88/CD40 demonstrated increased CAR MFI compared to the MC.ζ format(FIG. 27). This indicated that removing MC from the CAR domain restoredCAR stability or mitigated CAR-dependent MyD88/CD40 toxicity.

To examine whether constitutive expression of MyD88/CD40 could providecostimulation following CAR engagement to tumor cells bearing thecognate antigen, coculture experiments were performed usingCD19-targeted CARs with CD19+ Raji lymphoma cells. Here, MyD88/CD40,whether it was expressed within the CAR molecule itself, or as aconstitutive protein, enabled T cells to secrete IL-2 and IL-6 whichrequire costimulation in addition to CD3ζ signaling (FIG. 28). Furtheranalysis of these coculture assays using flow cytometry revealed thatwhile all CD19-specific CAR constructs efficiently killed Raji tumorcells (FIG. 29), only MC.ζ and 2A.MC CAR formats allowed T cellproliferation in response to antigenic stimulation (FIG. 30). These datasuggest that constitutive costimulation with MyD88/CD40 can preservehigh CAR expression levels while simultaneously providing signaling forT cell proliferation and survival.

In addition to CD19-targeted CARs, similar experiments were performedexamining whether Her2-specific CARs with the alternative MyD88/CD40format would function as with the CD19-specific CARs. As with the CD19CARs, constitutively expressing MyD88/CD40 by a 2A element improved CARMFI compared to Her2.MC. (FIGS. 31 and 32), and enhanced cytokineproduction (FIG. 33). Next, coculture assays were conducted against theHer2+ SK-BR-3 breast cancer cell line, and both Her2.MC.ζ as well as2A.MC demonstrated enhanced tumor control and corresponding T cellproliferation, consistent with potent costimulation (FIGS. 34 and 35).

To evaluate the antitumor potency of the 2A format, tumor xenograftanimal studies were performed. Immune deficient NSG mice were engraftedwith SK-BR-3-EGFPluciferase tumor cells and after 7 days, treated with 2doses of 1×10⁷T cells that were either non-transduced (NT), ortransduced with Her2.ζ. Her2.28.ζ. Her2.MC.ζ or Her2.ζ.2A.MC viaintratumoral injection. Mice treated with Her2.ζ.2A.MC-modified T cellsshowed complete tumor regression by day 14 post-T cell injection (FIG.36). Importantly, T cell toxicity as characterized by weight loss wasnot observed. These data further support that MyD88/CD40 can beconstitutively co-expressed with a CAR to support T cell growth andantitumor activity.

In summary, MyD88/CD40 can both be incorporated into a CAR molecule(scFv.MC.ζ), or as a constitutively expressed accessory protein, whichwhen introduced into primary T cells with a first generation CAR(scFv.ζ.2A.MC), enhances cytokine production, proliferation andantitumor activity both in vitro and in vivo.

Example 18: Nucleotide and Amino Acid Sequence ofpBP0813-SFG-iCasp9.2A.CD19.Zeta.2A.MC

Plasmid pBP0813-SFG-iCasp9.2A.CD19.zeta.2A.MC comprises a polynucleotideencoding an example of a chimeric antigen receptor of the presenttechnology; this polynucleotide does not include a membrane-targetingregion. The polynucleotide also encodes a chimeric inducible Caspase-9polypeptide.

FKBP12v36 ATGGGAGTGCAGGTGGAGACTATTAGCCCCGGAGATGGCAGAACATTCCCCAAAAGAGGACAGACTTGCGTCGTGCATTATACTGGAATGCTGGAAGACGGCAAGAAGGTGGACAGCAGCCGGGACCGAAACAAGCCCTTCAAGTTCATGCTGGGGAAGCAGGAAGTGATCCGGGGCTGGGAGGAAGGAGTCGCACAGATGTCAGTGGGACAGAGGGCCAAACTGACTATTAGCCCAGACTACGCTTATGGAGCAACCGGCCACCCCGGGATCATTCCCCCTCATGCTACACTGGTCTTCGATGTGGAGCTGCTGAAGCTGGAAMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE Linker AGCGGAGGAGGATCCGGASGGGSG Caspase-9GTGGACGGGTTTGGAGATGTGGGAGCCCTGGAATCCCTGCGGGGCAATGCCGATCTGGCTTACATCCTGTCTATGGAGCCTTGCGGCCACTGTCTGATCATTAACAATGTGAACTTCTGCAGAGAGAGCGGGCTGCGGACCAGAACAGGATCCAATATTGACTGTGAAAAGCTGCGGAGAAGGTTCTCTAGTCTGCACTTTATGGTCGAGGTGAAAGGCGATCTGACCGCTAAGAAAATGGTGCTGGCCCTGCTGGAACTGGCTCGGCAGGACCATGGGGCACTGGATTGCTGCGTGGTCGTGATCCTGAGTCACGGCTGCCAGGCTTCACATCTGCAGTTCCCTGGGGCAGTCTATGGAACTGACGGCTGTCCAGTCAGCGTGGAGAAGATCGTGAACATCTTCAACGGCACCTCTTGCCCAAGTCTGGGCGGGAAGCCCAAACTGTTCTTTATTCAGGCCTGTGGAGGCGAGCAGAAAGATCACGGCTTCGAAGTGGCTAGCACCTCCCCCGAGGACGAATCACCTGGAAGCAACCCTGAGCCAGATGCAACCCCCTTCCAGGAAGGCCTGAGGACATTTGACCAGCTGGATGCCATCTCAAGCCTGCCCACACCTTCTGACATTTTCGTCTCTTACAGTACTTTCCCTGGATTTGTGAGCTGGCGCGATCCAAAGTCAGGCAGCTGGTACGTGGAGACACTGGACGATATCTTTGAGCAGTGGGCCCATTCTGAAGACCTGCAGAGTCTGCTGCTGCGAGTGGCCAATGCTGTCTCTGTGAAGGGGATCTACAAACAGATGCCAGGATGCTTCAACTTTCTGAGAAAGAAACTGTTCTTTAAGACCTCCGCATCTAGGGCCVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSASRA Linker CCGCGG PR T2AGAAGGCCGAGGGAGCCTGCTGACATGTGGCGATGTGGAGGAAAACCCAGGACCAEGRGSLLTCGDVEENPGP Linker CCATGG PW Signal peptideATGGAGTTTGGACTTTCTTGGTTGTTTTTGGTGGCAATTCTGAAGGGTGTCCAGTGTAGCAGGMEFGLSWLFLVAILKGVQCSR FMC63 scFv V_(L)_GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAA TAACADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT LinkerGGCGGAGGAAGCGGAGGTGGGGGC GGGSGGGG FMC63 scFv V_(H)GAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAATTCAGCTCTCAAATCCAGACTGACCATCATCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS Linker GGATCC GSCD34 QBEND-10 epitope GAACTTCCTACTCAGGGGACTTTCTCAAACGTTAGCACAAACGTAAGTELPTQGTFSNVSTNVS CD8 alpha stalkCCCGCCCCAAGACCCCCCACACCTGCGCCGACCATTGCTTCTCAACCCCTGAGTTTGAGACCCGAGGCCTGCCGGCCAGCTGCCGGCGGGGCCGTGCATACAAGAGGACTCGATTTCGCTTGC GACPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8 alpha transmembraneATCTATATCTGGGCACCTCTCGCTGGCACCTGTGGAGTCCTTCTGCTCAGCCTGGTTATTACTCTGTACTGTAATCACCGGAATCGCCGCCGCGTTTGTAAGTGTCCCAGGIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR Linker GTCGAC VD CD3 zetaAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAAGCTCTTCCACCTCGTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR P2AGCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCTATNFSLLKQAGDVEENPGP Myristoylation sequenceatggggagtagcaagagcaagcctaaggaccccagccagcgc MGSSKSKPKDPSQR Linker ctcgacLD MyD88atggctgcaggaggtcccggcgcggggtctgcggccccggtctcctccacatcctcccttcccctggctgctctcaacatgcgagtgcggcgccgcctgtctctgttcttgaacgtgcggacacaggtggcggccgactggaccgcgctggcggaggagatggactttgagtacttggagatccggcaactggagacacaagcggaccccactggcaggctgctggacgcctggcagggacgccctggcgcctctgtaggccgactgctcgatctgcttaccaagctgggccgcgacgacgtgctgctggagctgggacccagcattgaggaggattgccaaaagtatatcttgaagcagcagcaggaggaggctgagaagcctttacaggtggccgctgtagacagcagtgtcccacggacagcagagctggcgggcatcaccacacttgatgaccccctggggcatatgcctgagcgtttcgatgccttcatctgctattgccccagcgacatcMAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFEYLEIRQLETQADPTGRLLDAWQGRPGASVGRLLDLLTKLGRDDVLLELGPSIEEDCQKYILKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI Linker gtcgag VE CD40aaaaaggtggccaagaagccaaccaataaggccccccaccccaagcaggagccccaggagatcaattttcccgacgatcttcctggctccaacactgctgctccagtgcaggagactttacatggatgccaaccggtcacccaggaggatggcaaagagagtcgcatctcagtgcaggagagacagKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

Example 19: MyD88/CD40-Based Costimulation to Enhance Survival andProliferation of Chimeric Antigen Receptor (CAR)-Modified T Cells

The efficacy of therapy with chimeric antigen receptor (CAR) T cells isassociated with T cell expansion, persistence, and elaboration ofmultiple cytokines, in response to antigen exposure following in vivoadoptive transfer. Chimeric stimulating molecules including MyD88, CD40,or MyD88/CD40 polypeptides were assayed for their ability to costimulateCAR T cell activity. Cells that also co-expressed an inducible chimericCaspase-9 polypeptide were assayed for their effectiveness as a safetyswitch; administration of the inducing ligand resulted in normalizationof cytokine levels without loss of tumor control in in vivo tumormodels.

Chimeric antigen receptor molecules that contain costimulatory domainssuch as CD28 and CD137 (4-1BB) exhibit varying degrees of persistenceand proliferation, but have uniformly shown limited anti-tumor effectswhen used to treat solid tumors. Rather than including a CD28 or 4-1 BBcostimulatory domain as part of the CAR molecule, a chimericcostimulating molecule fusion costimulatory molecule comprised of MyD88and CD40 (MyD88/CD40; “MC”) activates broad costimulatory pathways(e.g., NF-κB, MAPK, Akt, JNK) in human T cells that can driveproliferation and survival when expressed in CAR-T cells. To ensure thesafety of highly potent MC-enabled CARs, an iCaspase-9 safety switch(iC9) was been incorporated to allow either complete or partialelimination of CAR T cells through titration of the small moleculedimerizing agent, rimiducid (AP1903) This safety switch rapidly clears Tcells and reduces cytokine levels following rimiducid infusion.Titrating rimiducid, allowed for partial T cell elimination that stillpreserved CAR-T cell function.

Retrovirus and Transduction

CAR molecules containing single chain variable fragments (scFv) specificfor CD19 (FMC63) and Her2 (FRPS) containing the anti-CD34 QBEnd-10minimal epitope, CD8 stalk and transmembrane region and the CD3ζcytoplasmic domain were cloned in-frame with the inducible chimericCaspase-9-encoding polynucleotide. Additional constructs were made thatincluded the CD28 costimulatory domain, or MyD88, CD40 or MyD88/CD40(FIG. 37). T cells were transduced with EGFPluciferase (enhanced greenfluorescent protein luciferase) to monitor in vivo expansion. PBMCs wereactivated with αCD3ζ and αCD28 antibodies and transduced withretrovirus. After 10 days, transduction was measured using CD3ζ, CD19and CD34 antibodies.

Coculture Assays:

T cells transduced with CAR vectors were cocultured together withGFP-expressing CD19⁺ Raji lymphoma or Her2⁺ SK-BR-3 breast cancer celllines in the absence of exogenous IL-2. Cytokine production was assessedat 48 hours using ELISAs. Tumor and T cell number was measured usingflow cytometry and cell counting on day 10.

Animal Models:

CD19:

5×10⁵ Raji tumor cells were injected i.v. into NSG mice. On day 3, CD19CAR-modified T cells were injected i.v. and bioluminescence (BLI) wasmeasured for either tumor or T cells on a weekly basis by IVIS imaging.Mice losing >20% body weight were treated i.p. with 5 mg/kg rimiducid.

ic9 Titration:

NSG mice were engrafted with Raji tumors, as above, then administeredi.v. 5×10⁶ CAR-modified T cells. After 15% body weight loss, mice weretreated with rimiducid i.p. using a log-dose titration (0.0005-5 mg/kg).

Her2:

1×10⁶ SK-BR-3 tumor cells were injected s.c. into NSG mice. After 7days, mice were treated with i.t. injection of CAR-modified T cells.Tumor growth was measured by calipers (2-3 days) and BLI (weekly). Tcell expansion BLI was measured by IVIS.

MyD88/CD40 (MC) Costimulation in CAR-T Cells

FIG. 38 provides results of assays of MC costimulation in Her2 CAR-Tcells. A) Transduction and detection of primary T cells withiCasp9-Her2.ζ-MC CAR construct. B) IL-2 production from a cocultureexperiment mixing CAR-T cells with Her2⁺ SK-BR-3-GFP tumor cells (1:1ratio) after 48 hours (n=4). C and D) T cell and SK-BR-3-GFP tumor cellnumber from coculture experiment with different CAR constructs following10 days of culture. E) Efficacy of tumor cell elimination comparingCAR-T cell costimulation with CD40 only, MyD88 only or with MC in acoculture assay against SK-BR-3-GFP after 10 days in culture. F) IL-2production from coculture assay using constructs with the indicatedcostimulatory domains. *P-value=<0.01.

MC Enhances Her2 CAR-T Cell Efficacy In Vivo

FIG. 39 provides results of mouse in vivo efficacy assays using thevarious Her-2-specific CAR constructs. A) T cells were transduced withHer2.ζ, Her2.28.ζ or Her2.ζ-MC CARs and injected directly intoluciferase-expressing s.c. Her2+ SK-BR-3 tumors engrafted into NSG mice(n=5). Bioluminescence (BLI) of tumor cells was measured by IVIS. Tumorsize (B) was measured by calipers and survival (C) was calculated over75 days. D) T cells were subsequently co-transduced with CAR andluciferase and injected directly into s.c. Her2⁺ SK-BR-3 tumorsengrafted into NSG mice (n=5). E) CAR-T cell expansion was calculated byregion-of-interest ROI using IVIS imaging. F) Tumor size was calculatedby caliper measurements and shows individual mice in each treatmentgroup. *P-value=<0.05.

MC Enhances CD19 CAR-T Cell Efficacy In Vivo

FIG. 40 provides the results of mouse in vivo efficacy assays using thevarious CD19-specific CAR constructs. A) NSG mice (n=5 per group) wereengrafted with Raji-luciferase tumor cells and then treated withnon-transduced (NT) or iC9-CD19.ζ-MC CAR-modified T cells on day 3.Tumor growth was measured by IVIS imaging and calculated by whole-bodyBLI (B). C) Kaplan-Meier analysis from (A). At objective evidence ofsCRS, rimiducid was administered (red boxes, panel A), leading tonormalization of cytokines within 24 hrs and complete resolution ofclinical sCRS without compromising tumor control (not shown).

Titration of Inducible Chimeric Caspase-9 Safety Switch Enabled CARswith Rimiducid

FIG. 41 provides the results of mouse in vivo assays includingCD19-specific CAR constructs and the Caspase-9 safety switch. A and B)NSG mice (n=5 per group) were engrafted with CD19⁺ Raji lymphoma cellsand treated with 5×10⁶ iC9-CD19.ζ-MC/luciferase-transduced T cells atday 3. After 6 days, mice were treated i.p. with log dilutions ofrimiducid (0.00005-5 mg/kg). BLI of CAR-T cells was assessed prior torimiducid treatment and at 24 and 48 hours post-injection. C) Serumcytokine levels were measured from each group before (black line) and 24hours post-administration (orange line) of rimiducid. *P-value=<0.01.

Summary

In these assays, it was found that MyD88 and CD40 (“MC”) synergize toprovide potent costimulation in CAR-modified T cells targeting bothCD19+ “liquid” and Her2+“solid” tumors. The MC costimulation resulted inincreased T cell proliferation, cytokine production and antitumorefficacy in vivo compared to control CARs that included standardcostimulatory molecules (e.g., CD28). The constructs that also expressedthe inducible chimeric Caspase-9 polypeptide allowed for cessation oftherapy at high levels, and also combined a versatile, titratable, celltherapy safety switch with the MC-driven CAR T cells, permittingrimiducid-dependent normalization of cytokine levels without loss oftumor control in in vivo tumor models.

Example 20: MyD88/CD40 Chimeric Stimulating Molecule Activity in T CellsTransduced with Composite Retroviral Vectors

The signaling activity and physical expression of the chimericstimulating molecule MyD88/CD40 (MC) either in cytoplasmic for (MC) ormembrane-targeted form (myr-MC, for example was compared, and alsocompared to the inducible MyD88/CD40 molecule that includes amultimerizing ligand binding FKBP12 region. High level expression of MCwas sufficient to generate a substantial basal activity whether 5′ or 3′to DNA sequences encoding a Chimeric Antigen Receptor (CAR). Membranelocalization strongly induced signaling activity but may reducesteady-state MC protein expression. The FKBP12 fusion on the 3′ side ofMC attenuated basal MC activity, and dimerization with the FKBP ligandstrongly induced signaling activity without increasing iMC expression.

Methods Summary of DNA Constructs

The recombinant DNA vectors used to generate retroviruses capable oftransducing genes encoding a CAR and/or MC and/or inducible Caspase-9(iC9) as an operon are outlined schematically in FIGS. 48 and 49. Eachconstruct used the pSFG retroviral vector backbone:

pBP0844-pSFG-iCasp9-T2A-CD19-ζ-P2A-MC

This construct encodes human Caspase-9 fused with an SGGGSG linker 3′ toan F36V mutants of human FKBP12 with a short 5′ MLEMLE linker. A T2Acotranslational cleavage sequence derived from Thosea asigna virusseparates a sequence coding for an inducible chimeric caspasepolypeptide (iC9) from a chimeric antigen receptor (CAR) containing asingle chain variable fragment (scFv) targeting CD19, fused with a hingeand transmembrane domain, further fused on its cytoplasmic domain withthe chain of the T cell receptor CD3 complex. A P2A cotranslationalcleavage sequence derived from porcine teschovirus-1 virus separates theCAR from the human MyD88/CD40 (MC) fusion protein.

pBP0414-pSFG-iCasp9-T2A-CD19-ζ is identical to pBP0844 but does notinclude the P2A and MC sequences 3′ to the CAR.

pBP1099-pSFG-CD19-ζ encodes the CD19 CAR with its 5′ translationalinitiation site modified to match that of the plasmids discussed below.It served as a negative control for MC function.

pBP1151-pSFG-MC-T2A-CD19-ζ-P2A encodes MC (human MyD88-CD40 fusion) 5′to the CAR construct with the two polypeptides separated by the T2Acotranslational cleavage site.

pBP1152-pSFG-MyrMC-T2A-CD19-ζ-P2A encodes MC with a 5′myristoylation-targeting sequence derived from human c-Src. N-terminalmyristoylation of MC is predicted to lead to the accumulation of thesignaling molecule at the plasma membrane of transduced cells. The CARsequences are identical to pBP1151.

pBP0774-pSFG-iMC-T2A-CD19-ζ-P2A encodes MC as the soluble version of MCas a carboxy-terminal fusion with two tandem copies of human FKBP12v36,rendering MC rimiducid-inducible (iMC).

Costimulatory Activity Generated by MC Expression Constructs

HEK-293T cells were transduced with the SFG-based recombinant retroviralconstructs outlined above, with helper plasmids pBP0049 and pBP0175encoding the gag-pol and env genes necessary to package the recombinantRNAs as retroviruses. These retroviruses were transduced intoCD3/CD28-activated donor-derived primary T cells. Cytokine productionfrom transduced T cells was then used to assess the degree ofcostimulatory signaling activity conferred by the MC allele (if present)in the construct.

Three days following transduction, T cells were split and one populationtreated with 2 nM rimiducid. 24 or 48 hours after drug treatment, analiquot of media supernatant was harvested and the T cell-derivedcytokines, IL-2 and IL-6, were quantitated by enzyme linkedimmunosorbent assay (ELISA). IL-2 production typically requires bothsignal transduction from the antigen receptor (signal 1) via the NF-ATpathway and from costimulatory signals most simply assessed by NF-κBactivation. In this experiment, signal 1 was provided by the initialactivation of the T cells. It was found that T cells transduced withconstructs pBP1099 or pBP0414 supported little IL-2 production nor did Tcells that were not transduced at all (but were initially activated)(FIG. 50). In contrast, cells carrying pBP0844 had measurable IL-2production 24 hours after media from T cells was changed. Rimiducidtreatment only slightly reduced IL-2 production from these cells at 24hours. By 48 hours, IL-2 production was substantial at above 2 ng/mLfrom 1×10⁶ cells/mL. This ‘basal’ activity was dramatically lowered byrimiducid-mediated activation of apoptosis with iCaspase-9 at 48 hours.

Transduction with BP1151 (MC-CAR), encoding a comparable MC molecule butexpressed at the 5′ end of the bicistronic message, also had significantbasal IL-2 production at 24 and 48 hours. Rimiducid treatment did notaffect production significantly, as iC9 was not contained in thisconstruct. BP1152 (MyrMC-CAR) transduction supported very robust IL-2production, markedly elevated over BP1151 (MC-CAR), again withoutrespect to rimiducid treatment. This construct contained amyristoylated- and hence membrane-localized MC, which likely elevatedits signaling potential.

Markedly different basal activity was observed when cells weretransduced with iMC-encoding BP774. ‘Basal activity’ (IL-2 secretionwithout rimiducid) was minimal but rimiducid-mediated MC aggregationrevealed robust signaling as seen by high-level IL-2 production.

The inflammatory cytokine, IL-6, requires persistent costimulatorysignaling (signal 2) but had a reduced requirement for antigenreceptor-mediated NF-AT activity. IL-6 production is an independentassessment of MC activity in transduced cells. Overall, MC expressionhad a similar effect on IL-6 production, as it did with IL-2 levels intransduced human T cells. IL-6 secretion was negligible in the absenceof MC transduction (FIG. 51, untransduced cells or transduced withBP1099 or BP0414). Substantial ‘basal’ production was observed at 24 or48 hours from cells transduced with BP0844, which was silenceddramatically when iCaspase9 (iC9) was activated by rimiducid. Althoughsubstantial IL-6 production was induced by the non-targeted MC constructencoded by BP1151 (MC-CAR), IL-6 was highly elevated above these levelswhen MC contained the myristoylation-targeting domain (BP1152)(MyrMC-CAR). In both MC versions, which lack FKBPs, IL-6 production wasrimiducid-independent, as expected. A low, but detectable basalcostimulatory activity (˜110 μg/ml from 10⁶/mL T cells), consistent withbasal MC function, was observed with BP0774 transduction. Dimerizationwith 2 nM rimiducid robustly activated MC activity and IL-6 secretion toa level similar to that driven by the inducible chimeric Caspase-9construct BP844.

Summary

This example provides data demonstrating that MC chimeric costimulatingmolecules support significant cytokine production. The high cytokinerelease from T cells transduced with BP1152 (MyrMC-CAR) did notcorrelate with high-level steady-state protein expression. Localizationto the membrane via myristate tagging is likely to greatly enhance MCsignaling. It is possible that full Myr-MC expression may not beobserved due to incomplete solubilization of membrane proteins duringextract preparation. Because MyD88 and CD40 are naturally situated atthe plasma membrane, factors controlling their degradation may alsolocalize to the membrane leading to reduced Myr-MC expression. Thereduced observed expression of Myr-MC may also be due to high MCactivity being selected against in individual cells within a transducedpopulation. pBP1152 (MyrMC-CAR) had a reduced observed transductionefficiency and overall cell viability. Cells that were transducedexpressed less of the same CAR than cells transduced with BP1151(MC-CAR) possibly indicating selection for lower recombinant geneexpression in BP1152 (MyrMC-CAR). The source of the proposed selectionagainst high expression may be activation induced cell death (AICD) of Tcells. High-level MC signaling may possibly feed back and negativelyregulate MC protein expression. Myr-MC has high activity and low proteinexpression. Furthermore, T cells transduced with iMC encoded by BP0774had reduced expression of soluble iMC with rimiducid treatment despitefar greater MC activity. The reduction of ‘basal’ MC activity innon-localized iMC versus MC appeared to be greater than the more modestreduction of protein expression observed when comparing BP1151 (MC-CAR)and BP0774-transduced cells. FKBP12 fusion appeared to negatively affectspontaneous MC activity.

Example 21: Representative Embodiments

Provided hereafter are examples of certain embodiments of thetechnology.

A1. A nucleic acid comprising a polynucleotide encoding a chimericantigen receptor, wherein the chimeric antigen receptor comprises (i) atransmembrane region; (ii) a MyD88 polypeptide or a truncated MyD88polypeptide lacking a TIR domain; (iii) a CD40 cytoplasmic polypeptideregion lacking a CD40 extracellular domain; (iv) a T cell activationmolecule; and (v) an antigen recognition moiety.A1.1. The nucleic acid of embodiment A1, wherein the chimeric antigenreceptor further comprises a stalk polypeptide.A2. The nucleic acid of any of embodiments A1-A1.1, wherein the chimericantigen receptor is a polypeptide which comprises regions (i)-(v) inorder from the amino to the carboxyl terminal of the polypeptide of (v),(i), (ii), (iii), (iv).A3. The nucleic acid of any of embodiments A1-A1.1, wherein the chimericantigen receptor is a polypeptide which comprises regions (i)-(v) inorder from the amino to the carboxyl terminal of the polypeptide of (v),(i), (iii), (ii), (iv).

A4. Reserved. A5. Reserved.

A6. The nucleic acid of any one of embodiments A1-A3, wherein the T cellactivation molecule is an ITAM-containing, Signal 1 conferring molecule.A7. The nucleic acid of any one of embodiments A1-A6, wherein the T cellactivation molecule is a CD3ζ polypeptide.A8. The nucleic acid of any one of embodiments A1-A6, wherein the T cellactivation molecule is an Fc epsilon receptor gamma (FcεR1γ) subunitpolypeptide.A9. The nucleic acid of any one of embodiments A1-A8, wherein theantigen recognition moiety binds to an antigen on a tumor cell.A10. The nucleic acid of any one of embodiments A1-A9, wherein theantigen recognition moiety binds to an antigen on a cell involved in ahyperproliferative disease.A11. The nucleic acid of any one of embodiments A1-A10, wherein theantigen recognition moiety binds to an antigen selected from the groupconsisting of PSMA, PSCA, MUC1, CD19, ROR1, Mesothelin, GD2, CD123,MUC16, and Her2/Neu.A12. The nucleic acid of any one of embodiments A1-A11, wherein theantigen recognition moiety binds to PSCA.A13. The nucleic acid of any one of embodiments A1-A11, wherein theantigen recognition moiety binds to CD19.A14. The nucleic acid of any one of embodiments A1-A11, wherein theantigen recognition moiety binds to a viral or bacterial antigen.A15. The nucleic acid of any one of embodiments A1-A14, wherein theantigen recognition moiety is a single chain variable fragment.A16. The nucleic acid of any one of embodiments A1-A16, wherein thetransmembrane region is a CD8 transmembrane region.A17. The nucleic acid of any one of embodiments A1-A17, wherein theMyD88 polypeptide has the amino acid sequence of SEQ ID NO: 282, or afunctional fragment thereof.A18. The nucleic acid of any one of embodiments A1-A17, wherein thetruncated MyD88 polypeptide has the amino acid sequence of SEQ ID NO:147, or a functional fragment thereof.A19. The nucleic acid of any one of embodiments A1-A18, wherein thecytoplasmic CD40 polypeptide has the amino acid sequence of SEQ ID NO:149, or a functional fragment thereof.A20. The nucleic acid of any one of embodiments A1-A19, wherein theantigen recognition moiety is a single chain variable fragment thatbinds to CD19.A20.1. The nucleic acid of any one of embodiments A1-A19, wherein theantigen recognition moiety is a single chain variable fragment thatbinds to Her2/Neu.A21. The nucleic acid of any one of embodiments A1-A20.1, wherein theCD3ζ polypeptide has comprises an amino acid sequence of SEQ ID NO:151,or a functional fragment thereof.A22. The nucleic acid of any one of embodiments A1-A21, wherein thetransmembrane region polypeptide comprises an amino acid sequence of SEQID NO: 143, or a functional fragment thereof.

A23. Reserved. A24. Reserved.

A25. The nucleic acid of any one of embodiments A1-A24, wherein thenucleic acid comprises a promoter sequence operably linked to thepolynucleotide.A26. The nucleic acid of any one of embodiments A1-A25, wherein thenucleic acid is contained within a viral vector.A27. The nucleic acid of embodiment A26, wherein the viral vector is aretroviral vector.A28. The nucleic acid of embodiment A27, wherein the retroviral vectoris a murine leukemia virus vector.A29. The nucleic acid of embodiment A28, wherein the retroviral vectoris an SFG vector.A30. The nucleic acid of embodiment A26, wherein the viral vector is anadenoviral vector.A31. The nucleic acid of embodiment A26, wherein the viral vector is alentiviral vector.A31.1. The nucleic acid of embodiment A26, wherein the viral vector isselected from the group consisting of adeno-associated virus (AAV),Herpes virus, and Vaccinia virus.A31.2. The nucleic acid of any one of embodiments A1-A25, wherein thenucleic acid is prepared or in a vector designed for electroporation,sonoporation, or biolistics, or is attached to or incorporated inchemical lipids, polymers, inorganic nanoparticles, or polyplexes.A32. The nucleic acid of any one of embodiments A1-A25, wherein thenucleic acid is contained within a plasmid.A32.1. The nucleic acid of any one of embodiments A1-A32, comprising anucleotide sequence of Example 18, or encoding a chimeric antigenreceptor polypeptide of Example 18.A33. A chimeric antigen receptor polypeptide encoded by the nucleic acidof any one of embodiments A1-A32.1A34. A modified cell transfected or transduced with a nucleic acid ofany one of embodiments A1-A32.1.A34.1. The modified cell of embodiment A34, wherein the chimeric antigenreceptor does not contain a T cell activation molecule, furthercomprising a nucleic acid comprising a polynucleotide encoding a T cellactivation molecule.A34.2. The modified cell of embodiment A34.1, wherein the T cellactivation molecule is a CD3ζ polypeptide.A35. The modified cell of any one of embodiments A34-A34.2, wherein themodified cell is a T cell, tumor infiltrating lymphocyte, NK-T cell, orNK cell.A36. The modified cell of any one of embodiments A34-A34.2, wherein thecell is a T cell.A37. The modified cell of any one of embodiments A34-A36, wherein thecell is obtained or prepared from bone marrow.A38. The modified cell of any one of embodiments A34-A36, wherein thecell is obtained or prepared from umbilical cord blood.A39. The modified cell of any one of embodiments A34-A36, wherein thecell is obtained or prepared from peripheral blood.A40. The modified cell of any one of embodiments A34-A36, wherein thecell is obtained or prepared from peripheral blood mononuclear cells.A42. The modified cell of any one of embodiments A34-A40, wherein thecell is a human cell.A42.1. The modified cell of any one of embodiments A34-A42, wherein thecell is transfected or transduced by the nucleic acid vector using amethod selected from the group consisting of electroporation,sonoporation, biolistics (e.g., Gene Gun with Au-particles), lipidtransfection, polymer transfection, nanoparticles, or polyplexes.A43. A method for stimulating a cell mediated immune response to atarget cell population or tissue in a subject, comprising administeringa modified cell of any one of embodiments A34-A42.1 to the subject,wherein the antigen recognition moiety binds to an antigen on the targetcell.A44. The method of embodiment A43, wherein the target cell is a tumorcell.A45. The method of any one of embodiments A43 or A44, wherein the numberor concentration of target cells in the subject is reduced followingadministration of the modified cell.A46. The method of any one of embodiments A43-A45, comprising measuringthe number or concentration of target cells in a first sample obtainedfrom the subject before administering the modified cell, measuring thenumber concentration of target cells in a second sample obtained fromthe subject after administration of the modified cell, and determiningan increase or decrease of the number or concentration of target cellsin the second sample compared to the number or concentration of targetcells in the first sample.A47. The method of embodiment A46, wherein the concentration of targetcells in the second sample is decreased compared to the concentration oftarget cells in the first sample.A48. The method of embodiment A46, wherein the concentration of targetcells in the second sample is increased compared to the concentrationtarget cells in the first sample.A49. The method of any one of embodiments A43-A48, wherein an additionaldose of modified cells is administered to the subject.A50. A method for providing anti-tumor immunity to a subject, comprisingadministering to the subject an effective amount of a modified cell ofany one of embodiments A34-A42.1.A51. A method for treating a subject having a disease or conditionassociated with an elevated expression of a target antigen, comprisingadministering to the subject an effective amount of a modified cell ofany one of embodiments A34-A42.1.A52. The method of embodiment A51, wherein the target antigen is a tumorantigen.A53. The method of any one of embodiments A43-A52, wherein the modifiedcells are autologous T cells.A54. The method of any one of embodiments A43-A52, wherein the modifiedcells are allogeneic T cells.A55. A method for reducing the size of a tumor in a subject, comprisingadministering a modified cell of any one of embodiments A34-A42.1 to thesubject, wherein the antigen recognition moiety binds to an antigen onthe tumor.A56. The method of any one of embodiments A43-A55, wherein the subjecthas been diagnosed as having a tumor.A57. The method of any one of embodiments A43-A56, wherein the subjecthas cancer.A58. The method of any one of embodiments A43-A57, wherein the subjecthas a solid tumor.A59. The method of any one of embodiments A43-A58, wherein the modifiedcell is a tumor infiltrating lymphocyte or a T cell.A60. The method of any one of embodiments A43-A59, wherein the modifiedcell is delivered to a tumor bed.A61. The method of embodiment A57, wherein the cancer is present in theblood or bone marrow of the subject.A62. The method of any one of embodiments A43-A55, wherein the subjecthas a blood or bone marrow disease.A63. The method of any one of embodiments A43-A55, wherein the subjecthas been diagnosed with any condition or condition that can bealleviated by stem cell transplantation.A64. The method of any one of embodiments A43-A55, wherein the subjecthas been diagnosed with sickle cell anemia or metachromaticleukodystrophy.A65. The method of any one of embodiments A43-A55, wherein the patienthas been diagnosed with a condition selected from the group consistingof a primary immune deficiency condition, hemophagocytosislymphohistiocytosis (HLH) or other hemophagocytic condition, aninherited marrow failure condition, a hemoglobinopathy, a metaboliccondition, and an osteoclast condition.A66. The method of any one of embodiments A43-A65, wherein the diseaseor condition is selected from the group consisting of Severe CombinedImmune Deficiency (SCID), Combined Immune Deficiency (CID), CongenitalT-cell Defect/Deficiency, Common Variable Immune Deficiency (CVID),Chronic Granulomatous Disease, IPEX (Immune deficiency,polyendocrinopathy, enteropathy, X-linked) or IPEX-like, Wiskott-AldrichSyndrome, CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCA 8Deficiency, IL-10 Deficiency/IL-10 Receptor Deficiency, GATA 2deficiency, X-linked lymphoproliferative disease (XLP), Cartilage HairHypoplasia, Shwachman Diamond Syndrome, Diamond Blackfan Anemia,Dyskeratosis Congenita, Fanconi Anemia, Congenital Neutropenia, SickleCell Disease, Thalassemia, Mucopolysaccharidosis, Sphingolipidoses, andOsteopetrosis.A67. The method of any one of embodiments A43-A66, further comprisingdetermining whether an additional dose of the modified cell should beadministered to the subject.A68. The method of any one of embodiments A44-A67, further comprisingadministering an additional dose of the modified cell to the subject,wherein the disease or condition symptoms remain or are detectedfollowing a reduction in symptoms.A69. The method of any one of embodiments A44-A67, further comprisingidentifying the presence, absence or stage of a condition or disease ina subject; and

-   -   transmitting an indication to administer modified cell of any        one of embodiments 28-35, maintain a subsequent dosage of the        modified cell, or adjust a subsequent dosage of the modified        cell administered to the patient based on the presence, absence        or stage of the condition or disease identified in the subject.        A70. The method of any one of embodiments A44-A69, wherein the        condition is leukemia.        A71. The method of any one of embodiments A44-A70, wherein the        subject has been diagnosed with an infection of viral etiology        selected from the group consisting HIV, influenza, Herpes, viral        hepatitis, Epstein Bar, polio, viral encephalitis, measles,        chicken pox, Cytomegalovirus (CMV), adenovirus (ADV), HHV-6        (human herpesvirus 6, I), and Papilloma virus, or has been        diagnosed with an infection of bacterial etiology selected from        the group consisting of pneumonia, tuberculosis, and syphilis,        or has been diagnosed with an infection of parasitic etiology        selected from the group consisting of malaria, trypanosomiasis,        leishmaniasis, trichomoniasis, and amoebiasis.        A72. The method of any one of embodiments A44-A71, wherein the        modified cell is transfected or transduced in vivo.        A73. The modified cell of any one of embodiments A34-A42,        wherein the modified cell is transfected or transduced in vivo.        A74. A method for expressing a chimeric antigen receptor in a        cell, comprising contacting a nucleic acid of any one of        embodiments A1 to A33 with a cell under conditions in which the        nucleic acid is incorporated into the cell, whereby the cell        expresses the chimeric antigen receptor from the incorporated        nucleic acid.        A75. The method of embodiment A74, wherein the nucleic acid is        contacted with the cell ex vivo.        A76. The method of embodiment A74, wherein the nucleic acid is        contacted with the cell in vivo.        A77. The modified cell of any one of embodiments A34-A42,        wherein the modified cell further comprises a polynucleotide        encoding a chimeric Caspase-9 polypeptide comprising a        multimeric ligand binding region and a Caspase-9 polypeptide.        A77.1. A nucleic acid comprising    -   a first polynucleotide encoding a chimeric antigen receptor,        wherein the chimeric antigen receptor comprises (i) a        transmembrane region; (ii) a MyD88 polypeptide or a truncated        MyD88 polypeptide lacking a TIR domain; (iii) a CD40 cytoplasmic        polypeptide region lacking a CD40 extracellular domain; (iv) a T        cell activation molecule; and (v) an antigen recognition moiety;        and    -   a second polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide.        A77.2. The nucleic acid of embodiment A77.1, further comprising        at least one promoter.        A77.3. The nucleic acid of embodiment A77.1, further comprising        at least two promoters.        A77.4. The nucleic acid of embodiment A77.1, wherein one        promoter is operably linked to both the first and second        polynucleotide.        A77.5. The nucleic acid of embodiment A77.1, further comprising        a third polynucleotide encoding a linker polypeptide between the        first and second polynucleotide, wherein the linker polypeptide        separates the translation products of the first and second        polynucleotides during or after translation.        A77.6. The nucleic acid of embodiment A77.5, wherein the linker        polypeptide is a 2A polypeptide.        A77.7. The nucleic acid of embodiment A77.5, wherein the nucleic        acid encodes a polypeptide comprising a chimeric antigen        receptor, a 2A polypeptide, and a Caspase-9 polypeptide.        A77.8. The nucleic acid of embodiment A77.3, therein the first        polynucleotide is operably linked to a first promoter, and the        second polynucleotide is operably linked to a second promoter.        A77.9. The nucleic acid of embodiment A77.2, wherein two RNA        transcripts are produced complementary to the two        polynucleotides.        A77.10. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments A77.1-A77.9.        A78. The modified cell of any one of embodiments A77 or A77.10,        wherein the multimeric ligand binding region is an FKB12v36        region.        A79. The modified cell of any one of embodiments 77-78, wherein        the multimeric ligand is AP1903, or AP20187.        A80. The modified cell of any one of embodiments 77-79, wherein        the Caspase-9 polypeptide is a modified Caspase-9 polypeptide        comprising an amino acid substitution selected from the group        consisting of the caspase variants in Table 1.        A81. The method of any one of embodiments A44-A71, wherein the        modified cell further comprises a polynucleotide encoding a        chimeric caspase polypeptide comprising a multimeric ligand        binding region and a Caspase-9 polypeptide.        A82. The method of embodiment A81, wherein the multimeric ligand        binding region is an FKB12v36 region.        A83. The method of any one of embodiments A81-A82, wherein the        multimeric ligand is AP1903 or AP20187.        A84. The method of any one of embodiments A81-A83, wherein the        Caspase-9 polypeptide is a modified Caspase-9 polypeptide        comprising an amino acid substitution selected from the group        consisting of the caspase variants in Table 1.        A85. The method of any one of embodiments A81-A84, further        comprising administering the multimeric ligand to the subject        following administration of the modified cells to the subject.        A86. The method of embodiment A85, wherein after administration        of the multimeric ligand, the number of modified cells        comprising the chimeric Caspase-9 polypeptide is reduced.        A87. The method of embodiment A86, wherein the number of        modified cells comprising the chimeric Caspase-9 polypeptide is        reduced by 90%.        A88. The method of any one of embodiments A81-A87, comprising        determining that the subject is experiencing a negative symptom        following administration of the modified cells to the subject,        and administering the ligand to reduce or alleviate the negative        symptom.        B1. A nucleic acid comprising a polynucleotide encoding a        chimeric stimulating molecule, wherein the chimeric stimulating        molecule comprises (i) a membrane targeting region; (ii) a MyD88        polypeptide or a truncated MyD88 polypeptide lacking the TIR        domain; and (iii) a CD40 cytoplasmic polypeptide region lacking        the CD40 extracellular domain.        B1.1. A nucleic acid comprising a polynucleotide encoding a        chimeric stimulating molecule, wherein the chimeric stimulating        molecule comprises (i) a membrane targeting region; and (ii) a        MyD88 polypeptide or a truncated MyD88 polypeptide lacking the        TIR domain.        B1.2. A nucleic acid comprising a polynucleotide encoding a        chimeric stimulating molecule, wherein the chimeric stimulating        molecule comprises (i) a membrane targeting region; and (ii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain.        B2. The nucleic acid of any one of embodiments B1-B1.2, wherein        the chimeric stimulating molecule is a polypeptide which        comprises regions (i)-(iii) in order from the amino to the        carboxyl terminal of the polypeptide of (i), (ii), (iii).        B3. The nucleic acid of any one of embodiments B1-B1.2, wherein        the chimeric antigen receptor is a polypeptide which comprises        regions (i)-(iii) in order from the amino to the carboxyl        terminal of the polypeptide of (i), (iii), (ii).        B4. The nucleic acid of any one of embodiments B1-B3, wherein        the chimeric stimulating molecule further comprises a T cell        activation molecule.        B5. The nucleic acid of embodiment B4, wherein the T cell        activation molecule is an ITAM-containing signal 1 conferring        molecule.        B6. The nucleic acid of embodiment B4, wherein the T cell        activation molecule is a CD3ζ polypeptide.        B6.1. The nucleic acid of embodiment B4, wherein the T cell        activation molecule is an Fc epsilon receptor gamma (FcεR1γ)        subunit polypeptide.        B7. The nucleic acid of any one of embodiments B1-B6, wherein        the truncated MyD88 polypeptide has the amino acid sequence of        SEQ ID NO: 147, or a functional fragment thereof.        B7.1. The nucleic acid of any one of embodiments A1-A17, wherein        the MyD88 polypeptide has the amino acid sequence of SEQ ID NO:        282, or a functional fragment thereof.        B8. The nucleic acid of any one of embodiments B1, or B2-B7,        wherein the cytoplasmic CD40 polypeptide has the amino acid        sequence of SEQ ID NO: 149, or a functional fragment thereof.        B9. The nucleic acid of any one of embodiments B6-B8, wherein        the CD3ζ polypeptide comprises an amino acid sequence of SEQ ID        NO: 151, or a functional fragment thereof.        B10. The nucleic acid of any one of embodiments B1-B9, wherein        the membrane targeting region is selected from the group        consisting of a myristoylation region, palmitoylation region,        prenylation region, and transmembrane sequences of receptors.        B11. The nucleic acid of any one of embodiments B1-B10, wherein        the membrane targeting region is a myristoylation region.        B11.1. The nucleic acid of any one of embodiments B1-B10,        wherein the polynucleotide encoding the chimeric stimulating        molecule does not include a dimerization or multimerization        molecule binding region.        B12. The nucleic acid of any one of embodiments B1-B11.1,        wherein the nucleic acid comprises a promoter sequence operably        linked to the polynucleotide.        B13. The nucleic acid of any one of embodiments B1-B12, wherein        the nucleic acid is contained within a viral vector.        B14. The nucleic acid of embodiment B13, wherein the viral        vector is a retroviral vector.        B15. The nucleic acid of embodiment B14, wherein the retroviral        vector is a murine leukemia virus vector.        B16. The nucleic acid of embodiment B14, wherein the retroviral        vector is an SFG vector.        B17. The nucleic acid of embodiment B13, wherein the viral        vector is an adenoviral vector.        B18. The nucleic acid of embodiment B13, wherein the viral        vector is a lentiviral vector.        B18.1. The nucleic acid of embodiment B13, wherein the viral        vector is selected from the group consisting of adeno-associated        virus (AAV), Herpes virus, and Vaccinia virus.        B19. The nucleic acid of any one of embodiments B1-B12, wherein        the nucleic acid is contained within a plasmid.        B20. A chimeric stimulating molecule polypeptide encoded by the        nucleic acid of any one of embodiments B1-B19.        B21. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments B1-B19.        B22. The modified cell of embodiment B21, wherein the modified        cell is a T cell, tumor infiltrating lymphocyte, NK-T cell,        TCR-expressing cell, or NK cell.        B23. The modified cell of embodiment B21, wherein the cell is a        T cell.        B24. The modified cell of any one of embodiments B21-B23,        wherein the cell is obtained or prepared from bone marrow.        B25. The modified cell of any one of embodiments B21-B23,        wherein the cell is obtained or prepared from umbilical cord        blood.        B26. The modified cell of any one of embodiments B21-B25,        wherein the cell is obtained or prepared from peripheral blood.        B27. The modified cell of any one of embodiments B21-B25,        wherein the cell is obtained or prepared from peripheral blood        mononuclear cells.        B27.1 A modified cell comprising    -   a) a nucleic acid, wherein the nucleic acid comprises a        polynucleotide encoding a chimeric stimulating molecule, wherein        the chimeric stimulating molecule comprises (i) a membrane        targeting region; (ii) a MyD88 polypeptide or a truncated MyD88        polypeptide lacking the TIR domain; and (iii) a CD40 cytoplasmic        polypeptide region lacking the CD40 extracellular domain; and    -   b) a chimeric antigen receptor.        B27.2. A modified cell comprising    -   a) a nucleic acid, wherein the nucleic acid comprises a        polynucleotide encoding a chimeric stimulating molecule, wherein        the chimeric stimulating molecule comprises (i) a membrane        targeting region; and (ii) a MyD88 polypeptide or a truncated        MyD88 polypeptide lacking the TIR domain; and    -   b) a chimeric antigen receptor.        B27.3. A modified cell comprising    -   a) a nucleic acid, wherein the nucleic acid comprises a        polynucleotide encoding a chimeric stimulating molecule, wherein        the chimeric stimulating molecule comprises (i) a membrane        targeting region; and (ii) a CD40 cytoplasmic polypeptide region        lacking the CD40 extracellular domain; and    -   b) a chimeric antigen receptor.        B28. The modified cell of any one of embodiments B21-B27.3,        wherein the cell is a human cell.        B29. The modified cell of any one of embodiments B21-B28,        wherein the modified cell further comprises a polynucleotide        encoding a chimeric antigen receptor.        B30. The modified cell of embodiment B29, wherein the chimeric        antigen receptor comprises an antigen-recognition moiety.        B30.1. The modified cell of any one of embodiments B21-B30,        wherein the cell is a T cell.        B30.2. The modified cell of any one of embodiments B21-B28,        wherein the modified cell further comprises a polynucleotide        encoding a T cell receptor.        B30.3. The modified cell of any one of embodiments B21-28,        wherein the modified cell further comprises a polynucleotide        encoding a T cell receptor-based CAR.        B30.4. The modified cell of any one of embodiments B30.2 or        B30.3, wherein modified cell is transfected or transduced with a        nucleic acid comprising a polynucleotide encoding the T cell        receptor or T cell receptor-based CAR.        B31. The modified cell of any one of embodiments B27.1 or B30,        wherein the antigen-recognition moiety is a single chain        variable fragment.        B31.1. The modified cell of any one of embodiments B29-B31,        wherein the chimeric antigen receptor or T cell receptor binds        to an antigen on a tumor cell.        B32. The modified cell of any one of embodiments B29-B31.1,        wherein the chimeric antigen receptor or T cell receptor binds        to an antigen on a cell involved in a hyperproliferative        disease.        B33. The modified cell of any one of embodiments B29-B31.1,        wherein the chimeric antigen receptor or T cell receptor binds        to an antigen selected from the group consisting of PSMA, PSCA,        MUC1, CD19, ROR1, Mesothelin, GD2, CD123, MUC16, and Her2/Neu.        B34 The modified cell of any one of embodiments B29-B33, wherein        the chimeric antigen receptor or T cell receptor binds to CD19.        B35. The modified cell of any one of embodiments B29-B33,        wherein the chimeric antigen receptor or T cell receptor binds        to Her2/Neu.        B36. The modified cell of any one of embodiments B29-B33,        wherein the antigen recognition moiety binds to a viral or        bacterial antigen.        B36.1. The modified cell of any one of embodiments B29-B36,        wherein the cell is transfected or transduced by the nucleic        acid vector using a method selected from the group consisting of        electroporation, sonoporation, biolistics (e.g., Gene Gun with        Au-particles), lipid transfection, polymer transfection,        nanoparticles, or polyplexes.        B37. A method for stimulating a T cell-mediated immune response        in a subject, comprising administering a modified cell of any        one of embodiments B21-636.1 to the subject.        B37.1. The method of embodiment B37, wherein the modified cell        comprises a chimeric antigen receptor or T cell receptor that        binds to an antigen on a target cell.        B38. The method of embodiment B37.1, wherein the target cell is        a tumor cell.        B39. The method of any one of embodiments B37-B38, wherein the        number or concentration of target cells in the subject is        reduced following administration of the modified cell.        B40. The method of any one of embodiments B37-B39, comprising        measuring the number or concentration of target cells in a first        sample obtained from the subject before administering the        modified cell, measuring the number concentration of target        cells in a second sample obtained from the subject after        administration of the modified cell, and determining an increase        or decrease of the number or concentration of target cells in        the second sample compared to the number or concentration of        target cells in the first sample.        B41. The method of embodiment B40, wherein the concentration of        target cells in the second sample is decreased compared to the        concentration of target cells in the first sample.        B42. The method of embodiment B40, wherein the concentration of        target cells in the second sample is increased compared to the        concentration target cells in the first sample.        B43. The method of any one of embodiments B40-B42, wherein an        additional dose of modified cells is administered to the        subject.        B44. A method for providing anti-tumor immunity to a subject,        comprising administering to the subject an effective amount of a        modified cell of any one of embodiments B21-B36.1.        B45. A method for treating a subject having a disease or        condition associated with an elevated expression of a target        antigen, comprising administering to the subject an effective        amount of a modified cell of any one of embodiments B21-B36.1.        B46. The method of embodiment B45, wherein the target antigen is        a tumor antigen.        B47. The method of any one of embodiments B37-B46, wherein the        modified cells are autologous T cells.        B48. The method of any one of embodiments B37-B46, wherein the        modified cells are allogeneic T cells.        B50. A method for reducing the size of a tumor in a subject,        comprising administering a modified cell of any one of        embodiments B29-636.1 to the subject, wherein the antigen        recognition moiety binds to an antigen on the tumor.        B51. The method of any one of embodiments B37-B50, wherein the        subject has been diagnosed as having a tumor.        B52. The method of any one of embodiments B37-B51, wherein the        subject has cancer.        B53. The method of any one of embodiments B37-B51, wherein the        subject has a solid tumor.        B54. The method of any one of embodiments B37-B53, wherein the        modified cell is a tumor infiltrating lymphocyte or a T cell.        B55. The method of any one of embodiments B37-B54, wherein the        modified cell is delivered to a tumor bed.        B56. The method of embodiment B52, wherein the cancer is present        in the blood or bone marrow of the subject.        B57. The method of any one of embodiments B37-B51, wherein the        subject has a blood or bone marrow disease.        B58. The method of any one of embodiments B37-B51, wherein the        subject has been diagnosed with any condition or condition that        can be alleviated by stem cell transplantation.        B59. The method of any one of embodiments B37-B51, wherein the        subject has been diagnosed with sickle cell anemia or        metachromatic leukodystrophy.        B60. The method of any one of embodiments B37-B51, wherein the        patient has been diagnosed with a condition selected from the        group consisting of a primary immune deficiency condition,        hemophagocytosis lymphohistiocytosis (HLH) or other        hemophagocytic condition, an inherited marrow failure condition,        a hemoglobinopathy, a metabolic condition, and an osteoclast        condition.        B61. The method of any one of embodiments B37-B51, wherein the        disease or condition is selected from the group consisting of        Severe Combined Immune Deficiency (SCID), Combined Immune        Deficiency (CID), Congenital T-cell Defect/Deficiency, Common        Variable Immune Deficiency (CVID), Chronic Granulomatous        Disease, IPEX (Immune deficiency, polyendocrinopathy,        enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich Syndrome,        CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCA 8        Deficiency, IL-10 Deficiency/IL-10 Receptor Deficiency, GATA 2        deficiency, X-linked lymphoproliferative disease (XLP),        Cartilage Hair Hypoplasia, Shwachman Diamond Syndrome, Diamond        Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia,        Congenital Neutropenia, Sickle Cell Disease, Thalassemia,        Mucopolysaccharidosis, Sphingolipidoses, and Osteopetrosis.        B62. The method of any one of embodiments B37-B61, further        comprising determining whether an additional dose of the        modified cell should be administered to the subject.        B63. The method of any one of embodiments B37-B62, further        comprising administering an additional dose of the modified cell        to the subject, wherein the disease or condition symptoms remain        or are detected following a reduction in symptoms.        B64. The method of any one of embodiments B37-B63, further        comprising identifying the presence, absence or stage of a        condition or disease in a subject; and    -   transmitting an indication to administer modified cell of any        one of embodiments B31-B36, maintain a subsequent dosage of the        modified cell, or adjust a subsequent dosage of the modified        cell administered to the patient based on the presence, absence        or stage of the condition or disease identified in the subject.        B65. The method of any one of embodiments B37-B64, wherein the        condition is leukemia.

B66. Reserved.

B67. The method of any one of embodiments B37-B64, wherein the subjecthas been diagnosed with an infection of viral etiology selected from thegroup consisting HIV, influenza, Herpes, viral hepatitis, Epstein Bar,polio, viral encephalitis, measles, chicken pox, Cytomegalovirus (CMV),adenovirus (ADV), HHV-6 (human herpesvirus 6, I), and Papilloma virus,or has been diagnosed with an infection of bacterial etiology selectedfrom the group consisting of pneumonia, tuberculosis, and syphilis, orhas been diagnosed with an infection of parasitic etiology selected fromthe group consisting of malaria, trypanosomiasis, leishmaniasis,trichomoniasis, and amoebiasis.B68. The method of any one of embodiments B37-B67, wherein the modifiedcell is transfected or transduced in vivo.B69. The modified cell of any one of embodiments B21-B67, wherein themodified cell is transfected or transduced in vivo.B70. A method for expressing a chimeric stimulating molecule in a cell,comprising contacting a nucleic acid of any one of embodiments B1 to B20with a cell under conditions in which the nucleic acid is incorporatedinto the cell, whereby the cell expresses the chimeric antigen receptorfrom the incorporated nucleic acid.B71. The method of embodiment B70, wherein the nucleic acid is contactedwith the cell ex vivo.B72. The method of embodiment B70, wherein the nucleic acid is contactedwith the cell in vivo.B73. The modified cell of any one of embodiments B1-B36.1, wherein themodified cell further comprises a polynucleotide encoding a chimericCaspase-9 polypeptide comprising a multimeric ligand binding region anda Caspase-9 polypeptide.B73.1. A nucleic acid comprising

-   -   a first polynucleotide encoding a chimeric stimulating molecule,        wherein the chimeric stimulating molecule comprises (i) a        membrane targeting region; (ii) a MyD88 polypeptide or a        truncated MyD88 polypeptide lacking the TIR domain; and (iii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain; and    -   a second polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide.        B73.2. The nucleic acid of embodiment B73.1, further comprising        at least one promoter.        B73.3. The nucleic acid of embodiment B73.1, further comprising        at least two promoters.        B73.4. The nucleic acid of embodiment B73.1, wherein one        promoter is operably linked to both the first and second        polynucleotide.        B73.5. The nucleic acid of embodiment B73.1, further comprising        a third polynucleotide encoding a linker polypeptide between the        first and second polynucleotide, wherein the linker polypeptide        separates the translation products of the first and second        polynucleotides during or after translation.        B73.6. The nucleic acid of embodiment B73.5, wherein the linker        polypeptide is a 2A polypeptide.        B73.7. The nucleic acid of embodiment B73.5, wherein the nucleic        acid encodes a polypeptide comprising a chimeric stimulating        molecule, a 2A polypeptide, and a Caspase-9 polypeptide.        B73.8. The nucleic acid of embodiment B73.3, therein the first        polynucleotide is operably linked to a first promoter, and the        second polynucleotide is operably linked to a second promoter.        B73.9. The nucleic acid of embodiment B73.2, wherein two RNA        transcripts are produced complementary to the two        polynucleotides.        B73.10. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments B73.1-B73.9.        B74. The modified cell of any one of embodiments B73 or B73.10,        wherein the multimeric ligand binding region is an FKB12v36        region.        B75. The modified cell of any one of embodiments B73, B73.10, or        B74, wherein the multimeric ligand is AP1903 or AP20187.        B76. The modified cell of any one of embodiments B73-B75,        wherein the Caspase-9 polypeptide is a modified Caspase-9        polypeptide comprising an amino acid substitution selected from        the group consisting of the caspase variants in Table 1.        B77. The method of any one of embodiments B37-B72, wherein the        modified cell further comprises a polynucleotide encoding a        chimeric caspase polypeptide comprising a multimeric ligand        binding region and a Caspase-9 polypeptide.        B78. The method of embodiment B77, wherein the multimeric ligand        binding region is an FKB12v36 region.        B79. The method of any one of embodiments B77-B78, wherein the        multimeric ligand is AP1903, or AP20187.        B80. The method of any one of embodiments B77-B79, wherein the        Caspase-9 polypeptide is a modified Caspase-9 polypeptide        comprising an amino acid substitution selected from the group        consisting of the caspase variants in Table 1.        B81. The method of any one of embodiments B77-B80, further        comprising administering the multimeric ligand to the subject        following administration of the modified cells to the subject.        B82. The method of embodiment B81, wherein after administration        of the multimeric ligand, the number of modified cells        comprising the chimeric Caspase-9 polypeptide is reduced.        B83. The method of embodiment B82, wherein the number of        modified cells comprising the chimeric Caspase-9 polypeptide is        reduced by 90%.        B84. The method of any one of embodiments B77-B83, comprising        determining that the subject is experiencing a negative symptom        following administration of the modified cells to the subject,        and administering the ligand to reduce or alleviate the negative        symptom.        C1. A nucleic acid comprising a promoter operably linked to a        polynucleotide encoding a cytoplasmic chimeric stimulating        molecule, wherein the cytoplasmic chimeric stimulating molecule        comprises (i) a MyD88 polypeptide or a truncated MyD88        polypeptide lacking the TIR domain; and (ii) a CD40 cytoplasmic        polypeptide region lacking the CD40 extracellular domain.        C2. A nucleic acid comprising a promoter operably linked to a        polynucleotide encoding a chimeric stimulating molecule, wherein        the chimeric stimulating molecule comprises (i) a MyD88        polypeptide or a truncated MyD88 polypeptide lacking the TIR        domain; and (ii) a CD40 cytoplasmic polypeptide region lacking        the CD40 extracellular domain, with the proviso that the        chimeric stimulating molecule does not include a membrane        targeting region.        C3. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a cytoplasmic chimeric stimulating molecule, wherein the        cytoplasmic chimeric stimulating molecule comprises (i) a MyD88        polypeptide or a truncated MyD88 polypeptide lacking the TIR        domain; and (ii) a CD40 cytoplasmic polypeptide region lacking        the CD40 extracellular domain; and    -   b) a second polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide.        C4. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a chimeric stimulating molecule, wherein the chimeric        stimulating molecule comprises (i) a MyD88 polypeptide or a        truncated MyD88 polypeptide lacking the TIR domain; and (ii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain, with the proviso that the chimeric        stimulating molecule does not include a membrane targeting        region; and    -   b) a second polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide.        C5. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a cytoplasmic chimeric stimulating molecule, wherein the        cytoplasmic chimeric stimulating molecule comprises (i) a MyD88        polypeptide or a truncated MyD88 polypeptide lacking the TIR        domain; and (ii) a CD40 cytoplasmic polypeptide region lacking        the CD40 extracellular domain; and    -   b) a second polynucleotide encoding a chimeric antigen receptor.        C6. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a chimeric stimulating molecule, wherein the chimeric        stimulating molecule comprises (i) a MyD88 polypeptide or a        truncated MyD88 polypeptide lacking the TIR domain; and (ii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain, with the proviso that the chimeric        stimulating molecule does not include a membrane targeting        region; and    -   b) a second polynucleotide encoding a chimeric antigen receptor.        C7. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a cytoplasmic chimeric stimulating molecule, wherein the        cytoplasmic chimeric stimulating molecule comprises (i) a MyD88        polypeptide or a truncated MyD88 polypeptide lacking the TIR        domain; and (ii) a CD40 cytoplasmic polypeptide region lacking        the CD40 extracellular domain; and    -   b) a second polynucleotide encoding a T cell receptor or a T        cell receptor-based chimeric antigen receptor.        C8. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a chimeric stimulating molecule, wherein the chimeric        stimulating molecule comprises (i) a MyD88 polypeptide or a        truncated MyD88 polypeptide lacking the TIR domain; and (ii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain, with the proviso that the chimeric        stimulating molecule does not include a membrane targeting        region; and    -   b) a second polynucleotide encoding a T cell receptor or a T        cell receptor-based chimeric antigen receptor.        C9. A nucleic acid comprising    -   a) a promoter operably linked to a first polynucleotide encoding        a chimeric stimulating molecule, wherein the chimeric        stimulating molecule comprises (i) a MyD88 polypeptide or a        truncated MyD88 polypeptide lacking the TIR domain; and (ii) a        CD40 cytoplasmic polypeptide region lacking the CD40        extracellular domain, with the proviso that the chimeric        stimulating molecule does not include a membrane targeting        region; and    -   b) a second polynucleotide encoding a T cell receptor, a T cell        receptor-based chimeric antigen receptor, or a chimeric antigen        receptor; and    -   c) a third polynucleotide encoding a chimeric Caspase-9        polypeptide comprising a multimeric ligand binding region and a        Caspase-9 polypeptide.        C10. The nucleic acid of any one of embodiments C5-C6 or C8-C9,        wherein the chimeric antigen receptor comprises (i) a        transmembrane region; (ii) a T cell activation molecule;        and (iii) an antigen recognition moiety.        C11. The nucleic acid of embodiment C10, wherein the chimeric        antigen receptor further comprises a co-stimulatory molecule.        C12. The nucleic acid of embodiment C11, wherein the        co-stimulatory molecule is selected from the group consisting of        CD28, OX40, and 4-1BB.        C13. The nucleic acid of any one of embodiments C1-C12,        comprising a second promoter operably linked to the second        polynucleotide.        C14. The nucleic acid of embodiment C9, comprising a second        promoter operably linked to the second polynucleotide and a        third promoter operably linked to the third polynucleotide.        C15. The nucleic acid of any one of embodiments C1-C8, or        C10-C14, wherein one promoter is operably linked to both the        first and second polynucleotides.        C16. The nucleic acid of any one of embodiments C9, or C13-C14,        wherein one promoter is operably linked to the first, second,        and third polynucleotides.        C17. The nucleic acid of embodiment C15, further comprising a        third polynucleotide encoding a linker polypeptide between the        first and second polynucleotides, wherein the linker polypeptide        separates the translation products of the first and second        polynucleotides during or after translation.        C18. The nucleic acid of embodiment C16, further comprising        polynucleotides encoding linker polypeptides between the three        polynucleotides, wherein the three polynucleotides comprise the        first, second, and third polynucleotides, wherein the linker        polypeptides separate the translation products of the three        polynucleotides during or after translation,        C19. The nucleic acid of any one of embodiments C17 or C18,        wherein the linker polypeptide is a 2A polypeptide.        C20. The nucleic acid of any one of embodiments C10-C19, wherein        the T cell activation molecule is an ITAM-containing, Signal 1        conferring molecule.        C21. The nucleic acid of any one of embodiments C10-C19, wherein        the T cell activation molecule is a CD3ζ polypeptide.        C22. The nucleic acid of any one of embodiments C10-C19, wherein        the T cell activation molecule is an Fc epsilon receptor gamma        (FccR1γ) subunit polypeptide.        C23. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to an antigen on a tumor        cell.        C24. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to an antigen on a cell        involved in a hyperproliferative disease.        C25. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to an antigen selected from        the group consisting of PSMA, PSCA, MUC1, CD19, ROR1,        Mesothelin, GD2, CD123, MUC16, and Her2/Neu.        C26. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to PSCA.        C27. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to CD19.        C28. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to Her2/Neu.        C29. The nucleic acid of any one of embodiments C10-C22, wherein        the antigen recognition moiety binds to a viral or bacterial        antigen.        C30. The nucleic acid of any one of embodiments C10-C29, wherein        the antigen recognition moiety is a single chain variable        fragment.        C31. The nucleic acid of any one of embodiments C10-C30, wherein        the transmembrane region is a CD28 transmembrane region.        C32. The nucleic acid of any one of embodiments C10-C30, wherein        the transmembrane region is a CD8 transmembrane region.        C33. The nucleic acid of any one of embodiments C31-C32, wherein        the chimeric antigen receptor further comprises a CD8 stalk        region.        C33.1. The nucleic acid of any one of embodiments C1-C33, with        the proviso that the cytoplasmic chimeric stimulating molecule        does not include a multimeric ligand binding region.        C34. The nucleic acid of any one of embodiments C1-C33.1,        wherein the truncated MyD88 polypeptide has the amino acid        sequence of SEQ ID NO: 147, or a functional fragment thereof.        C35. The nucleic acid of any one of embodiments C1-C33.1,        wherein the MyD88 polypeptide has the amino acid sequence of SEQ        ID NO: 282, or a functional fragment thereof.        C36. The nucleic acid of any one of embodiments C1-C35, wherein        the cytoplasmic CD40 polypeptide has the amino acid sequence of        SEQ ID NO: 149, or a functional fragment thereof.        C37. The nucleic acid of any one of embodiments C21-C36, wherein        the CD3ζ polypeptide comprises an amino acid sequence of SEQ ID        NO: 151, or a functional fragment thereof.        C38. The nucleic acid of any one of embodiments C3-C4, or        C9-C37, wherein the multimeric ligand binding region is a ligand        binding region selected from the group consisting of FKBP,        cyclophilin receptor, steroid receptor, tetracycline receptor,        heavy chain antibody subunit, light chain antibody subunit,        single chain antibodies comprised of heavy and light chain        variable regions in tandem separated by a flexible linker        domain, and mutated sequences thereof.        C39. The nucleic acid of embodiment C38, wherein the ligand        binding region is an FKBP12 region.        C40. The nucleic acid of embodiment C39, wherein the FKBP12        region is an FKBP12v36 region. C41. The nucleic acid of        embodiment C38, wherein the FKBP region is Fv′Fvls.        C42. The nucleic acid of any one of embodiments C3-C4 or C9-C37,        wherein the ligand is an FK506 dimer or a dimeric FK506 analog        ligand.        C43. The nucleic acid of any one of embodiments C38-C42, wherein        the ligand is AP1903 or AP20187.        C44. The nucleic acid of any one of embodiments C1-C43, wherein        the nucleic acid is contained within a viral vector.        C45. The nucleic acid of embodiment C44, wherein the viral        vector is a retroviral vector.        C46. The nucleic acid of embodiment C45, wherein the retroviral        vector is a murine leukemia virus vector.        C47. The nucleic acid of embodiment C45, wherein the retroviral        vector is an SFG vector.        C48. The nucleic acid of embodiment C44, wherein the viral        vector is an adenoviral vector.        C48. The nucleic acid of embodiment C44, wherein the viral        vector is a lentiviral vector.        C50. The nucleic acid of embodiment C44, wherein the viral        vector is selected from the group consisting of adeno-associated        virus (AAV), Herpes virus, and Vaccinia virus.        C51. The nucleic acid of any one of embodiments C1-C43, wherein        the nucleic acid is contained within a plasmid.        C52. A chimeric stimulating molecule polypeptide encoded by the        nucleic acid of any one of embodiments C1-C2, or C34-C36.        D1. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments C1-C51.        D2. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments C1-C2, or C10-C51.        D3. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments C1-C2, or C10-C51, with the        proviso that the nucleic acid does not comprise a polynucleotide        encoding a chimeric antigen receptor and does not comprise a        polynucleotide encoding a chimeric Caspase-9 polypeptide.        D4. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments C1-C4, or C10-C51.        D5. A modified cell transfected or transduced with a nucleic        acid of any one of embodiments C1-C4, or C10-C51, with the        proviso that the nucleic acid does not comprise a polynucleotide        encoding a chimeric antigen receptor.        D5.1. The modified cell of any one of embodiments D1-D5, wherein        the cytoplasmic chimeric stimulating molecule is constitutively        expressed.        D5.2. The modified cell of any one of embodiments D1-D5.2,        wherein the cytoplasmic chimeric stimulating molecule is        constitutively active.        D6. The modified cell of embodiments D1-D5.2, wherein the        modified cell further comprises a nucleic acid comprising a        polynucleotide encoding a chimeric antigen receptor.        D7. The modified cell of embodiments D1-D3, wherein the modified        cell further comprises a nucleic acid comprising a        polynucleotide encoding a chimeric Caspase-9 polypeptide,        wherein the chimeric Caspase-9 polypeptide comprises a        multimeric ligand binding region and a Caspase-9 polypeptide.        D8. The modified cell of embodiment D6, wherein the chimeric        antigen receptor comprises (i) a transmembrane region; (ii) a T        cell activation molecule; and (iii) an antigen recognition        moiety.        D9. The modified cell of embodiment D8, wherein the chimeric        antigen receptor further comprises a co-stimulatory molecule.        D10. The modified cell of embodiment D9, wherein the        co-stimulatory molecule is selected from the group consisting of        CD28, OX40, and 4-1 BB.        D11. The modified cell of any one of embodiments D8-D10, wherein        the T cell activation molecule is an ITAM-containing, Signal 1        conferring molecule.        D12. The modified cell of any one of embodiments D8-D10, wherein        the T cell activation molecule is a CD3ζ polypeptide.        D13. The modified cell of any one of embodiments D8-D10, wherein        the T cell activation molecule is an Fc epsilon receptor gamma        (FcεR1γ) subunit polypeptide.        D14. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to an antigen on a tumor        cell.        D15. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to an antigen on a cell        involved in a hyperproliferative disease.        D16. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to an antigen selected from        the group consisting of PSMA, PSCA, MUC1, CD19, ROR1,        Mesothelin, GD2, CD123, MUC16, and Her2/Neu.        D17. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to PSCA.        D18. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to CD19.        D19. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to Her2/Neu.        D20. The modified cell of any one of embodiments D8-D13, wherein        the antigen recognition moiety binds to a viral or bacterial        antigen.        D21. The modified cell of any one of embodiments D8-D20, wherein        the antigen recognition moiety is a single chain variable        fragment.        D22. The modified cell of any one of embodiments D8-D21, wherein        the transmembrane region is a CD28 transmembrane region.        D23. The modified cell of any one of embodiments D8-D21, wherein        the transmembrane region is a CD8 transmembrane region.        D24. The modified cell of any one of embodiments D22-D23,        wherein the chimeric antigen receptor further comprises a CD8        stalk region.        D25. The modified cell of embodiment D7, wherein the multimeric        ligand binding region is a ligand binding region selected from        the group consisting of FKBP, cyclophilin receptor, steroid        receptor, tetracycline receptor, heavy chain antibody subunit,        light chain antibody subunit, single chain antibodies comprised        of heavy and light chain variable regions in tandem separated by        a flexible linker domain, and mutated sequences thereof.        D26. The modified cell of embodiment D25, wherein the ligand        binding region is an FKBP12 region.        D27. The modified cell of embodiment D26, wherein the FKBP12        region is an FKBP12v36 region.        D28. The modified cell of embodiment D25, wherein the FKBP        region is Fv′Fvls.        D29. The modified cell of any one of embodiments D25-D28,        wherein the ligand is an FK506 dimer or a dimeric FK506 analog        ligand.        D30. The modified cell of any one of embodiments D25-D28,        wherein the ligand is AP1903 or AP20187.        D31. The modified cell of any one of embodiments D1-D30, wherein        the modified cell is a T cell, tumor infiltrating lymphocyte,        NK-T cell, TCR-expressing cell, or NK cell.        D32. The modified cell of any one of embodiments D1-D30, wherein        the cell is a T cell.        D33. The modified cell of any one of embodiments D1-D30, wherein        the cell is obtained or prepared from bone marrow.        D34. The modified cell of any one of embodiments D1-D30, wherein        the cell is obtained or prepared from umbilical cord blood.        D35. The modified cell of any one of embodiments D1-D30, wherein        the cell is obtained or prepared from peripheral blood.        D36. The modified cell of any one of embodiments D1-D30, wherein        the cell is obtained or prepared from peripheral blood        mononuclear cells.        D37. The modified cell of any one of embodiments D1-D30, wherein        the cell is a human cell.        D38. The modified cell of any one of embodiments D1-D30, wherein        the cell is transfected or transduced by the nucleic acid vector        using a method selected from the group consisting of        electroporation, sonoporation, biolistics (e.g., Gene Gun with        Au-particles), lipid transfection, polymer transfection,        nanoparticles, or polyplexes.        E1. A method for stimulating a T cell-mediated immune response        in a subject, comprising administering an effective amount of        modified cells of any one of embodiments D1-D38 to the subject.        E2. The method of embodiment E1, wherein the modified cell        comprises a chimeric antigen receptor or T cell receptor that        binds to an antigen on a target cell.        E3. The method of embodiment E1, wherein the target cell is a        tumor cell.        E4. The method of any one of embodiments E2-E3, wherein the        number or concentration of target cells in the subject is        reduced following administration of the modified cell.        E5. The method of any one of embodiments E2-E4, comprising        measuring the number or concentration of target cells in a first        sample obtained from the subject before administering the        modified cell, measuring the number concentration of target        cells in a second sample obtained from the subject after        administration of the modified cell, and determining an increase        or decrease of the number or concentration of target cells in        the second sample compared to the number or concentration of        target cells in the first sample.        E6. The method of embodiment E5, wherein the concentration of        target cells in the second sample is decreased compared to the        concentration of target cells in the first sample.        E7. The method of embodiment E5, wherein the concentration of        target cells in the second sample is increased compared to the        concentration target cells in the first sample.        E8. The method of any one of embodiments E1-E7, wherein an        additional dose of modified cells is administered to the        subject.        E9. A method for providing anti-tumor immunity to a subject,        comprising administering to the subject an effective amount of a        modified cell of any one of embodiments E1-E9.        E10. A method for treating a subject having a disease or        condition associated with an elevated expression of a target        antigen, comprising administering to the subject an effective        amount of a modified cell of any one of embodiments D1-D38.        E11. The method of embodiment E10, wherein the target antigen is        a tumor antigen.        E12. A method for reducing the size of a tumor in a subject,        comprising administering a modified cell of any one of        embodiments D1-D38 to the subject, wherein the cell comprises a        chimeric antigen receptor or T cell receptor comprising an        antigen recognition moiety binds to an antigen on the tumor.        E13. The method of any one of embodiments E1-E12, wherein the        subject has been diagnosed as having a tumor.        E14. The method of any one of embodiments E1-E12, wherein the        subject has cancer.        E15. The method of any one of embodiments E1-E12, wherein the        subject has a solid tumor.        E16. The method of any one of embodiments E1-E12, wherein the        modified cell is a tumor infiltrating lymphocyte or a T cell.        E17. The method of any one of embodiments E1-E16, wherein the        modified cell is delivered to a tumor bed.        E18. The method of embodiment E14, wherein the cancer is present        in the blood or bone marrow of the subject.        E19. The method of any one of embodiments E10-E18, wherein the        subject has a blood or bone marrow disease.        E20. The method of any one of embodiments E10-E18, wherein the        subject has been diagnosed with any condition or condition that        can be alleviated by stem cell transplantation.        E21. The method of any one of embodiments E10-E18, wherein the        subject has been diagnosed with sickle cell anemia or        metachromatic leukodystrophy.        E22. The method of any one of embodiments E10-E18, wherein the        subject has been diagnosed with a condition selected from the        group consisting of a primary immune deficiency condition,        hemophagocytosis lymphohistiocytosis (HLH) or other        hemophagocytic condition, an inherited marrow failure condition,        a hemoglobinopathy, a metabolic condition, and an osteoclast        condition.        E23. The method of any one of embodiments E10-E18, wherein the        disease or condition is selected from the group consisting of        Severe Combined Immune Deficiency (SCID), Combined Immune        Deficiency (CID), Congenital T-cell Defect/Deficiency, Common        Variable Immune Deficiency (CVID), Chronic Granulomatous        Disease, IPEX (Immune deficiency, polyendocrinopathy,        enteropathy, X-linked) or IPEX-like, Wiskott-Aldrich Syndrome,        CD40 Ligand Deficiency, Leukocyte Adhesion Deficiency, DOCA 8        Deficiency, IL-10 Deficiency/IL-10 Receptor Deficiency, GATA 2        deficiency, X-linked lymphoproliferative disease (XLP),        Cartilage Hair Hypoplasia, Shwachman Diamond Syndrome, Diamond        Blackfan Anemia, Dyskeratosis Congenita, Fanconi Anemia,        Congenital Neutropenia, Sickle Cell Disease, Thalassemia,        Mucopolysaccharidosis, Sphingolipidoses, and Osteopetrosis.        E24. The method of any one of embodiments E1-E23, further        comprising determining whether an additional dose of the        modified cell should be administered to the subject.        E25. The method of any one of embodiments E1-E24, further        comprising administering an additional dose of the modified cell        to the subject, wherein the disease or condition symptoms remain        or are detected following a reduction in symptoms.        E26. The method of any one of embodiments E1-E25 further        comprising identifying the presence, absence or stage of a        condition or disease in a subject; and transmitting an        indication to administer modified cell of any one of embodiments        D1-D38, maintain a subsequent dosage of the modified cell, or        adjust a subsequent dosage of the modified cell administered to        the patient based on the presence, absence or stage of the        condition or disease identified in the subject.        E27. The method of any one of embodiments E10-E26, wherein the        condition is leukemia.        E28. The method of any one of embodiments E10-E26, wherein the        subject has been diagnosed with an infection of viral etiology        selected from the group consisting HIV, influenza, Herpes, viral        hepatitis, Epstein Bar, polio, viral encephalitis, measles,        chicken pox, Cytomegalovirus (CMV), adenovirus (ADV), HHV-6        (human herpesvirus 6, I), and Papilloma virus, or has been        diagnosed with an infection of bacterial etiology selected from        the group consisting of pneumonia, tuberculosis, and syphilis,        or has been diagnosed with an infection of parasitic etiology        selected from the group consisting of malaria, trypanosomiasis,        leishmaniasis, trichomoniasis, and amoebiasis.        E29. The method of any one of embodiments E1-E28, wherein the        modified cell comprises a chimeric Caspase-9 polypeptide        comprising a multimeric ligand binding region and a Caspase-9        polypeptide.        E30. The method of embodiment E29, further comprising        administering a multimeric ligand that binds to the multimeric        ligand binding region to the subject following administration of        the modified cells to the subject.        E31. The method of embodiment E30, wherein after administration        of the multimeric ligand, the number of modified cells        comprising the chimeric Caspase-9 polypeptide is reduced.        E32. The method of embodiment E31, wherein the number of        modified cells comprising the chimeric Caspase-9 polypeptide is        reduced by 50%.        E33. The method of embodiment E31, wherein the number of        modified cells comprising the chimeric Caspase-9 polypeptide is        reduced by 75%.        E34. The method of embodiment E31, wherein the number of        modified cells comprising the chimeric Caspase-9 polypeptide is        reduced by 90%.        E35. The method of any one of embodiments E29-E32, comprising        determining that the subject is experiencing a negative symptom        following administration of the modified cells to the subject,        and administering the ligand to reduce or alleviate the negative        symptom.        E36. The method of any one of embodiments E29-E35, wherein the        ligand is AP1903 or AP20187.        E37. The method of any one of embodiments E1-E36, wherein the        modified cells are autologous T cells.        E38. The method of any one of embodiments E1-E36, wherein the        modified cells are allogeneic T cells.        E39. The method of any one of embodiments E1-E38, wherein the        modified cells are transfected or transduced in vivo.        E40. The modified cell of any one of embodiments E1-E38, wherein        the modified cells are transfected or transduced ex vivo.        E41. A method for expressing a chimeric stimulating molecule in        a cell, comprising contacting a nucleic acid of any one of        embodiments C1-052 with a cell under conditions in which the        nucleic acid is incorporated into the cell, whereby the cell        expresses the chimeric antigen receptor from the incorporated        nucleic acid.        E42. The method of embodiment E41, wherein the nucleic acid is        contacted with the cell ex vivo.        E43. The method of embodiment E41, wherein the nucleic acid is        contacted with the cell in vivo.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the technologyclaimed. The term “a” or “an” can refer to one of or a plurality of theelements it modifies (e.g., “a reagent” can mean one or more reagents)unless it is contextually clear either one of the elements or more thanone of the elements is described. The term “about” as used herein refersto a value within 10% of the underlying parameter (i.e., plus or minus10%), and use of the term “about” at the beginning of a string of valuesmodifies each of the values (i.e., “about 1, 2 and 3” refers to about 1,about 2 and about 3). For example, a weight of “about 100 grams” caninclude weights between 90 grams and 110 grams. Further, when a listingof values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or86%) the listing includes all intermediate and fractional values thereof(e.g., 54%, 85.4%). Thus, it should be understood that although thepresent technology has been specifically disclosed by representativeembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and such modifications and variations are considered within thescope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

1-35. (canceled)
 36. A modified cell comprising a nucleic acidcomprising a first polynucleotide encoding a chimeric stimulatingmolecule, wherein the chimeric stimulating molecule comprises: (i) aMyD88 polypeptide or a truncated MyD88 polypeptide lacking the TIRdomain; (ii) a CD40 cytoplasmic polypeptide region lacking the CD40extracellular domain; and (iii) a membrane targeting region; wherein thechimeric stimulating molecule does not include a multimeric ligandbinding region, and wherein the chimeric stimulating molecule isconstitutively active.
 37. The modified cell of claim 36, wherein themembrane targeting region is selected from the group consisting of amyristoylation region, a palmitoylation region, a prenylation region,and a transmembrane sequences of receptors.
 38. The modified cell ofclaim 36, wherein the first polynucleotide is operably linked to apromoter.
 39. The modified cell of claim 36, wherein the nucleic acidfurther comprises a second polynucleotide encoding a chimeric antigenreceptor, comprising: (i) an scFv antigen recognition moiety; (ii) atransmembrane region; and (iii) a CD3 zeta polypeptide.
 40. The modifiedcell of claim 39, wherein the chimeric antigen receptor comprises a CD8stalk region between the scFv antigen recognition moiety and the CD8transmembrane region.
 41. The modified cell of claim 39, wherein thescFv antigen recognition moiety binds to an antigen selected from thegroup consisting of PSMA, PSCA, MUC1, CD19, ROR1, Mesothelin, GD2,CD123, MUC16, Her2/Neu, CD20, CD30, PRAME, NY-ESO-1, and EGFRvIII. 42.The modified cell of claim 39, wherein the scFv antigen recognitionmoiety binds to an antigen selected from the group consisting of PSCA,CD19, and Her2/Neu.
 43. The modified cell of claim 42, wherein the scFvantigen recognition moiety binds to Her2/Neu.
 44. The modified cell ofclaim 39, wherein the scFv antigen recognition moiety comprises theamino acid sequence of SEQ ID NO: 155 and the amino acid sequence of SEQID NO:
 159. 45. The modified cell of claim 42, wherein the scFv antigenrecognition moiety binds to CD19.
 46. The modified cell of claim 39,wherein the scFv antigen recognition moiety comprises the amino acidsequence of SEQ ID NO: 131 and the amino acid sequence of SEQ ID NO:135.
 47. The modified cell of claim 36, wherein the modified cell is a Tcell, a tumor infiltrating lymphocyte, a NK-T cell, a TCR-expressingcell, or a NK cell.
 48. The modified cell of claim 36, wherein themodified cell is a T cell.
 49. The modified cell of claim 36, whereinthe modified cell is a NK cell.
 50. The modified cell of claim 37,wherein the membrane targeting region is a myristoylation region. 51.The modified cell of claim 36, wherein the truncated MyD88 polypeptidehas the amino acid sequence of SEQ ID NO: 147, or a functional fragmentthereof.
 52. The modified cell of claim 36, wherein the MyD88polypeptide has the amino acid sequence of SEQ ID NO: 282, or afunctional fragment thereof.
 53. The modified cell of claim 36, whereinthe cytoplasmic CD40 polypeptide has the amino acid sequence of SEQ IDNO: 149, or a functional fragment thereof.
 54. The modified cell ofclaim 36, wherein the nucleic acid further comprises a thirdpolynucleotide encoding a chimeric Caspase-9 polypeptide comprising amultimeric ligand binding region and a Caspase-9 polypeptide.
 55. Themodified cell of claim 54, wherein the Caspase-9 polypeptide comprisesthe amino acid sequence of SEQ ID NO:
 153. 56. The modified cell ofclaim 54, wherein the multimeric ligand binding region is an FKBP12region.
 57. The modified cell of claim 56, wherein the FKBP12 region isan FKBP12v36 region.